Rna sequence adaptation

ABSTRACT

The present invention is directed to a method for modifying the retention time of RNA on a chromatographic column. The present invention also concerns a method for purifying RNA from a mixture of at least two RNA species. Furthermore, the present invention relates to a method for co-purifying at least two RNA species from a mixture of at least two RNA species. In particular, the present invention provides a method for harmonizing the numbers of A and/or U nucleotides in at least two RNA species. The present invention is also directed to RNA obtainable by said methods, a composition comprising said RNA or a vaccine comprising said RNA and methods for producing such RNA and compositions. Further, the invention concerns a kit, particularly a kit of parts, comprising the RNA, composition or vaccine. The invention is further directed to a method of treating or preventing a disorder or a disease, first and second medical uses of the RNA, composition and vaccine. Moreover, the present invention concerns a method for providing an adapted RNA sequence or an adapted RNA mixture.

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/EP2018/080692, filed Nov. 8, 2018,which claims benefit of International Application No. PCT/EP2017/078647,filed Nov. 8, 2017, the entire contents of each of which are herebyincorporated by reference.

INTRODUCTION

The present invention is directed to a method for modifying theretention time of RNA on a chromatographic column. The present inventionalso concerns a method for purifying RNA from a mixture of at least twoRNA species. Furthermore, the present invention relates to a method forco-purifying at least two RNA species from a mixture of at least two RNAspecies. In particular, the present invention provides a method forharmonizing the numbers of A and/or U nucleotides in at least two RNAspecies. The present invention is also directed to RNA obtainable bysaid methods, a composition comprising said RNA or a vaccine comprisingsaid RNA and methods for producing such RNA and compositions. Further,the invention concerns a kit, particularly a kit of parts, comprisingthe RNA, composition or vaccine. The invention is further directed to amethod of treating or preventing a disorder or a disease, first andsecond medical uses of the RNA, composition and vaccine. Moreover, thepresent invention concerns a method for providing an adapted RNAsequence or an adapted RNA mixture.

Ribonucleic acid (RNA)-based therapeutics can be used in immunotherapy,gene therapy and genetic vaccination. They can provide highly specificand individual treatment options for the therapy of a large variety ofdiseases.

For certain medical treatments and applications, it is desired to applyan RNA mixture comprising different RNA molecule species. Examples ofsuch treatments based on an RNA mixture include the application ofpolyvalent RNA mixtures that provide protection against severalserotypes of a pathogen (e.g. hemagglutinin (HA) from multiple serotypesof Influenza A and B virus); RNA mixtures that provide differentantigens from one pathogen (e.g. different antigens from Influenza, suchas HA, nucleoprotein (NP), neuraminidase (NA) etc.); RNA mixtures thatprovide protection against several isoforms or variants of a cancerantigen (e.g. prostate specific antigen (PSA) in the context of prostatecarcinoma); RNA mixtures that provide different epitopes of an antigen;RNA mixtures that contain a cancer specific and/or patient specificmixture of cancer antigens (expressed antigens or mutated antigens); RNAmixtures that encode a variety of antibodies (e.g., antibodies that aretargeted against different epitopes of one or more proteins), or anyother therapeutically active RNA mixture (e.g., encoding differentisoforms of an enzyme for molecular therapy, different therapeuticproteins for treatment of an indication wherein several proteins have tobe supplemented).

A significant step forward in the field of RNA-based therapeutics wasachieved by the establishment of an RNA manufacturing process that hasbeen approved by regulatory authorities, implementing various qualitycontrols on DNA level and RNA level as described in detail inWO2016/180430A1. A key element of said process is the preparativepurification of the RNA product via RP-HPLC (as described in WO2008/077592A1).

So far, RNA production methods are, however, only suitable to produceone single specific RNA molecule species at a time, such as an RNAmolecule encoding one specific therapeutic target. Each RNA moleculespecies has to be produced in a separate production process. For theproduction of an RNA mixture comprising different RNA molecule species,separate production processes have to be performed, and the separatelyproduced RNA molecule species have to be mixed to generate an RNAmixture. Accordingly, the conventional production of an RNA mixture(e.g. polyvalent vaccine) containing several RNA molecule species (e.g.RNA coding for different antigens, e.g. Influenza virus antigens) islaborious, costly, and time consuming since it requires several runs forDNA template production, RNA production and HPLC purification of the RNAproduct. Apart from economic reasons, especially in the context ofpandemic scenarios, an acceleration of the production of RNA mixtures(e.g. polyvalent RNA vaccine) may be highly advantageous and of majorimportance for public health. Therefore, it is desirable to produce andpurify RNA mixtures simultaneously, ideally in only one production andpurification process.

Simultaneous production, purification, and analysis of RNA mixtures areof particular importance for the manufacturing of multivalent/polyvalentvaccines, particularly a multivalent/polyvalent influenza RNA vaccine.Advantageously, the manufacturing of such a multivalent/polyvalentinfluenza RNA vaccine should allow for a fast exchange of (one or many)antigens (e.g. for seasonal adaptations of the vaccine; pandemicscenario) without changing the conditions for key processes ofproduction (e.g., purification, analysis etc.).

Recently, a procedure for the simultaneous production of different RNAmolecule species in one production process has been developed, asdescribed in WO20171090134, aiming to economize and accelerate theproduction of RNA mixture based therapeutics.

Simultaneous production of different RNA molecule species as e.g.described in WO20171090134 would save time, labor costs, productioncosts, and production capacities (e.g., space, equipment) in themanufacturing of RNA mixture based therapeutics. However, a severeproblem is associated with the use of RP-HPLC for simultaneouspreparative purification of the obtained RNA mixture (herein alsoreferred to as “co-purification”). Different RNA molecule species in anRNA mixture commonly elute at different time points (due to differentretention time in HPLC), which renders co-purification via HPLCtechnically impossible in most of the cases (illustrated in FIGS. 1A andB). For example, co-purification via HPLC would only be possible incases, where all different RNA molecule species comprised in the RNAmixture elute at essentially the same time point leading to an overlayof the different RNA product peaks facilitating separation of cleanfull-length RNA product mixture (peak fraction) and separation fromimpurities (illustrated in FIGS. 1C and D).

Accordingly, the purification of an RNA-mixture, such as an RNA-basedtherapeutic comprising multiple RNA species (e.g. a multivalent RNAvaccine), is not feasible with the current methodology known in the art.In particular, the co-purification of individual RNA species in an RNAmixture is not feasible. However, such a co-purification is highlydesirable, for example in the context of the development of amultivalent/polyvalent influenza RNA vaccine platform that is capable ofbeing adapted to seasonal changes in the antigen composition. In such asituation, rapid exchanges of antigens (e.g. influenza hemagglutinin(HA) and/or neuraminidase (NA)) are required. The methods available inthe field do not allow for the co-purification of different RNA speciesfrom an RNA mixture, in particular via HPLC, since different species ofRNA molecules encoding different antigens would typically elute atdifferent time points.

For similar reasons, also simultaneous HPLC-based quality control of RNAmixtures or (multivalent) vaccine (either produced in one productionprocess or individually produced and mixed) is not feasible when usingprior art techniques. For the analysis of therapeutics comprisingmixtures of RNAs, it is required that the different components (i.e.different RNA molecule species) of the drug product can be characterizedin terms of presence, integrity, ratio and quantity (quality controlparameter). Such quality controls may be implemented during or followingproduction of the RNA mixture, and/or as a batch release qualitycontrol. However, as described above, different species of RNA moleculesin an RNA mixture typically elute at different time points, whichstrongly impedes the simultaneous assessment of the quality/integrity ofthe individual RNA molecule species/or the whole RNA mixture via HPLC(“co-analysis”) (illustrated in FIGS. 1A and B). A reliabledetermination of the integrity of the RNA mixture via HPLC would only bepossible in cases where the clear discrimination of the peak areas foreach RNA molecule species is possible (simultaneous determination of theRNA quality for each RNA molecule species; see FIG. 1E) and/or wheredifferent RNA species in the RNA mixture elute at essentially the sametime point, leading to an essentially complete overlay of the HPLC peaks(determination of the RNA quality of the whole RNA mixture; see FIGS. 1Cand D).

Accordingly, also the analysis of an RNA mixture, such as an RNAtherapeutic comprising multiple RNA species (e.g. a multivalentInfluenza RNA vaccine), is not feasible with the current methodologyprovided in the art. In particular, co-analysis of RNA species in an RNAmixture is not possible. For example, co-analysis, e.g. via HPLC, is notapplicable in the case of a multivalent RNA vaccine platform, whererapid exchanges of antigens are required (e.g. for seasonal vaccines,such as an influenza vaccine), since different species of RNA moleculeseach encoding different antigens (such as Influenza HA and/or NA) eluteat different time points, typically leading to partially overlappingHPLC peaks.

In summary, the technical difficulties described above strongly hinderthe (co-)purification and the (co-)analysis of RNA mixtures. Inparticular, RP-HPLC, which represents a key element for the purificationand the analysis of RNA in general and particularly of GMP-grade RNA,cannot be employed in the (co-)purification and (co-)analysis ofindividual RNA species in RNA mixtures. Furthermore, the describedproblems have to be solved to facilitate the development of amultivalent/polyvalent RNA manufacturing process for producingfast-adjustable RNA vaccines, e.g. influenza vaccines

Summarizing the above, there remains an unmet need for a system thatallows for purification and/or analysis of RNA mixtures.

Therefore, it is the object of the underlying invention to provide asystem for purification and/or analysis of RNA, in particular of an RNAmixture. It is further a preferred object of the present invention toprovide a system for (co-)purification and/or (co-)analysis ofindividual RNA species in an RNA mixture comprising multiple RNAspecies.

This object is solved by the claimed subject matter.

DESCRIPTION OF THE INVENTION

The present application is filed together with a sequence listing inelectronic format, which is part of the description of the presentapplication. The information contained in the electronic format of thesequence listing filed together with this application is incorporatedherein by reference in its entirety. Where reference is made herein to a‘SEQ ID NO:’ the corresponding nucleic acid sequence or amino acidsequence in the sequence listing having the respective identifier isreferred to, unless stated otherwise.

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Definitions

Adaptive immune response: The term “adaptive immune response” as usedherein will be recognized and understood by the person of ordinary skillin the art, and is, for example, intended to refer to anantigen-specific response of the immune system. Antigen specificityallows for the generation of responses that are tailored to specificpathogens or pathogen-infected cells. The ability to mount thesetailored responses is usually maintained in the body by “memory cells”(B-cells). In the context of the invention, the antigen is provided bythe RNA coding region encoding at least one antigenic peptide, orprotein.

Adaptive immune system: The adaptive immune system is essentiallydedicated to eliminate or prevent pathogenic growth. It typicallyregulates the adaptive immune response by providing the vertebrateimmune system with the ability to recognize and remember specificpathogens (to generate immunity), and to mount stronger attacks eachtime the pathogen is encountered.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in thebroadest sense is typically a pharmacological and/or immunological agentthat may enhance the effect of other agents, such as a drug or vaccine.It is to be interpreted in a broad sense and refers to a broad spectrumof substances. Typically, these substances are able to increase theimmunogenicity of antigens. For example, adjuvants may be recognized bythe innate immune systems and, e.g., may elicit an innate immuneresponse. “Adjuvants” typically do not elicit an adaptive immuneresponse. Insofar, “adjuvants” do not qualify as antigens. Their mode ofaction is distinct from the effects triggered by antigens resulting inan adaptive immune response.

Antigen: In the context of the present invention “antigen” referstypically to a substance which may be recognized by the immune system,preferably by the adaptive immune system, and is capable of triggeringan antigen-specific immune response, e.g. by formation of antibodiesand/or antigen-specific T cells as part of an adaptive immune response.Typically, an antigen may be or may comprise a peptide or protein whichmay be presented by the MHC to T-cells. In the sense of the presentinvention an antigen may be the product of translation of a provided RNAas defined herein. In this context, also fragments, variants andderivatives of peptides and proteins comprising at least one epitope areunderstood as antigens. In the context of the present invention, tumorantigens and pathogenic antigens as defined herein are particularlypreferred.

Bicistronic RNA, multicistronic RNA: A bicistronic or multicistronic RNAis typically an RNA, preferably an mRNA, that typically may have two(bicistronic) or more (multicistronic) coding regions. A coding regionin this context is a sequence of codons that is translatable into apeptide or protein.

Carrier/polymeric carrier: A carrier in the context of the invention maytypically be a compound that facilitates transport and/or complexationof another compound (cargo). A polymeric carrier is typically a carrierthat is formed of a polymer. A carrier may be associated to its cargo bycovalent or non-covalent interaction. A carrier may transport nucleicacids, e.g. RNA or DNA, to the target cells. The carrier may—for someembodiments—be a cationic component.

Cationic component: The term “cationic component” typically refers to acharged molecule, which is positively charged (cation) at a pH valuetypically from 1 to 9, preferably at a pH value of or below 9 (e.g. from5 to 9), of or below 8 (e.g. from 5 to 8), of or below 7 (e.g. from 5 to7), most preferably at a physiological pH, e.g. from 7.3 to 7.4.Accordingly, a cationic component may be any positively charged compoundor polymer, preferably a cationic peptide or protein which is positivelycharged under physiological conditions, particularly under physiologicalconditions in vivo. A “cationic peptide or protein” may contain at leastone positively charged amino acid, or more than one positively chargedamino acid, e.g. selected from Arg, His, Lys or Orn. Accordingly,“polycationic” components are also within the scope exhibiting more thanone positive charge under the conditions given.

Coding region: A coding region, in the context of the invention, istypically a sequence of several nucleotide triplets, which may betranslated into a peptide or protein. A coding region preferablycontains a start codon, i.e. a combination of three subsequentnucleotides coding usually for the amino acid methionine (ATG), at its5′-end and a subsequent region which usually exhibits a length which isa multiple of 3 nucleotides. A coding region is preferably terminated bya stop-codon (e.g., TAA, TAG, TGA). Typically, this is the onlystop-codon of the coding region. Thus, a coding region in the context ofthe present invention is preferably a nucleotide sequence, consisting ofa number of nucleotides that may be divided by three, which starts witha start codon (e.g. ATG) and which preferably terminates with a stopcodon (e.g., TAA, TGA, or TAG). The coding region may be isolated or itmay be incorporated in a longer nucleic acid sequence, for example in avector or an mRNA. In the context of the present invention, a codingregion may also be termed “protein coding region”, “coding sequence”,“CDS”, “open reading frame” or “ORF”.

Fragment of a sequence: A fragment of a sequence may typically be ashorter portion of a full-length sequence of e.g. a nucleic acidmolecule or an amino acid sequence. Accordingly, a fragment, typically,comprises or consists of a sequence that is identical to thecorresponding stretch within the full-length sequence. A preferredfragment of a sequence in the context of the present invention,comprises or consists of a continuous stretch of entities, such asnucleotides or amino acids corresponding to a continuous stretch ofentities in the molecule the fragment is derived from, which representsat least 5%, 10%, 20%, preferably at least 30%, more preferably at least40%, more preferably at least 50%, even more preferably at least 60%,even more preferably at least 70%, and most preferably at least 80% ofthe total (i.e. full-length) molecule from which the fragment isderived. Preferably, a fragment of a sequence as used herein is at least5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, morepreferably at least 80%, even more preferably at least 90%, even morepreferably at least 95%, most preferably at least 99%, identical to asequence, from which it is derived. Preferably, a fragment as usedherein has the same biological function or specific activity compared tothe full-length molecule.

Heterologous sequence: Two sequences are typically understood to be‘heterologous’ if they are not derivable from the same gene or in thesame allele. I.e., although heterologous sequences may be derivable fromthe same organism, they naturally (in nature) do not occur in the samenucleic acid molecule, such as in the same mRNA.

Homolog (of a nucleic acid sequence/amino acid sequence): The term“homolog” typically refers to a sequence of the same or of anotherspecies that is related, but preferably not identical, to a referencesequence. The term “homolog” encompasses orthologs as well as paralogs.In this context, “orthologs” are proteins encoded by genes in differentspecies that evolved from a common ancestral gene by speciation.Orthologs often retain the same function(s) in the course of evolution.Thus, functions may be lost or gained when comparing a pair oforthologs. “Paralogs” are genes produced via gene duplication within agenome. Paralogs may also evolve new functions or eventually becomepseudogenes. In the context of the present invention, a homolog of anucleic acid sequence or of an amino acid sequence is preferably atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%,even more preferably at least 80%, even more preferably at least 90%,even more preferably at least 95%, most preferably at least 99%,identical to a reference sequence. It is further preferred that a“homolog” as used herein consists of a continuous stretch of entities,such as nucleotides or amino acid residues, corresponding to acontinuous stretch of entities in the reference molecule, whichrepresents at least 5%, 10%, 20%, preferably at least 30%, morepreferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, and mostpreferably at least 80% of the total (i.e. full-length) referencemolecule.

Immunogen: In the context of the present invention an immunogen may betypically understood to be a compound that is able to stimulate animmune response. Preferably, an immunogen is a peptide, polypeptide, orprotein. In a particularly preferred embodiment, an immunogen in thesense of the present invention is the product of translation of aprovided nucleic acid molecule, preferably an RNA molecule as definedherein. Typically, an immunogen elicits at least an adaptive immuneresponse.

Immune response: An immune response may typically be a specific reactionof the adaptive immune system to a particular antigen (so calledspecific or adaptive immune response) or an unspecific reaction of theinnate immune system (so called unspecific or innate immune response),or a combination thereof.

Immune system: The immune system may protect organisms from infection.If a pathogen succeeds in passing a physical barrier of an organism andenters this organism, the innate immune system provides an immediate,but non-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained after the pathogen has been eliminated, in the form of animmunological memory, and allows the adaptive immune system to mountfaster and stronger attacks each time this pathogen is encountered.According to this, the immune system comprises the innate and theadaptive immune system. Each of these two parts typically contains socalled humoral and cellular components.

Nucleic acid sequence/amino acid sequence: The sequence of a nucleicacid molecule is typically understood to be the particular andindividual order, i.e. the succession of its nucleotides. The sequenceof a protein or peptide is typically understood to be the order, i.e.the succession of its amino acid residues.

Peptide: A peptide or polypeptide is typically a polymer of amino acidmonomers, linked by peptide bonds. A peptide typically contains lessthan 50 monomer units. Nevertheless, the term peptide is not adisclaimer for molecules having more than 50 monomer units. Longpeptides are also called polypeptides, typically having between 50 and600 monomeric units. The term ‘polypeptide’ as used herein, however, istypically not limited by the length of the molecule it refers to. In thecontext of the present invention, the term ‘polypeptide’ may also beused with respect to peptides comprising less than 50 (e.g. 10) aminoacids or peptides comprising even more than 600 amino acids.

Polyvalent/polyvalent vaccine: A polyvalent vaccine (also referred to as‘multivalent vaccine’) typically contains antigens (or fragments orvariants thereof) from more than one strain of a virus, or differentantigens (or fragments or variants thereof) of the same virus, or anycombination thereof. The term “polyvalent vaccine” describes that thisvaccine has more than one valence. In the context of the invention, apolyvalent vaccine (e.g. Influenza vaccine, Norovirus vaccine)preferably comprises a vaccine comprising nucleic acids encodingantigenic peptides or proteins derived from several (genetically)different viruses or comprising nucleic acids encoding differentantigens from (genetically) the same virus, or a combination thereof. Ina preferred embodiment, a polyvalent vaccine comprises 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100 or even more different RNAs or RNA species, preferably asdescribed herein, each encoding at least one different antigenic peptideor protein. Methods to produce polyvalent mRNA vaccines are disclosed inthe PCT application WO20171090134.

Therapeutically effective amount: A therapeutically effective amount inthe context of the invention is typically understood to be an amountthat is sufficient to induce a pharmaceutical effect, such as an immuneresponse, altering a pathological level of an expressed peptide orprotein, or substituting a lacking gene product, e.g., in case of apathological situation.

Protein: A protein typically comprises one or more peptides orpolypeptides. A protein is typically folded into 3-dimensional form,which may be required for the protein to exert its biological function.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually adenosine-monophosphate, uridine-monophosphate,guanosine-monophosphate and cytidine-monophosphate monomers which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificsuccession of the monomers is called the RNA-sequence. Usually RNA maybe obtainable by transcription of a DNA-sequence, e.g., inside a cell.In eukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. Typically, transcription of DNA usuallyresults in the so-called premature RNA which has to be processed intoso-called messenger-RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional-modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino-acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap, a5′-UTR, a coding region, a 3′-UTR and a poly(A) sequence. Aside frommessenger RNA, several non-coding types of RNA exist which may beinvolved in regulation of transcription and/or translation.

Sequence identity, identity (of a sequence): Two or more sequences areidentical if they exhibit the same length and order of nucleotides oramino acids. The percentage of identity typically describes the extent,to which two sequences are identical, i.e. it typically describes thepercentage of nucleotides that correspond in their sequence positionwith identical nucleotides of a reference sequence. In order todetermine the degree of identity, the sequences to be compared areconsidered to exhibit the same length, i.e. the length of the longestsequence of the sequences to be compared. This means that a firstsequence consisting of 8 nucleotides is 80% identical to a secondsequence consisting of 10 nucleotides comprising the first sequence.Hence, in the context of the present invention, identity of sequencespreferably relates to the percentage of nucleotides of a sequence whichhave the same position in two or more sequences having the same length.Therefore, e.g. a position of a first sequence may be compared with thecorresponding position of the second sequence. If a position in thefirst sequence is occupied by the same component (residue) as is thecase at a position in the second sequence, the two sequences areidentical at this position. If this is not the case, the sequencesdiffer at this position. If insertions occur in the second sequence incomparison to the first sequence, gaps can be inserted into the firstsequence to allow a further alignment. If deletions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe second sequence to allow a further alignment. The percentage towhich two sequences are identical is then a function of the number ofidentical positions divided by the total number of positions includingthose positions which are only occupied in one sequence. The percentageto which two sequences are identical can be determined using amathematical algorithm. A preferred, but not limiting, example of amathematical algorithm which can be used is the algorithm of Karlin etal. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), NucleicAcids Res., 25:3389-3402. Such an algorithm is integrated in the BLASTprogram. Sequences which are identical to the sequences of the presentinvention to a certain extent can be identified by this program.

Transfection: The term “transfection” refers to the introduction ofnucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, intocells, preferably into eukaryotic cells. In the context of the presentinvention, the term “transfection” encompasses any method known to theskilled person for introducing nucleic acid molecules into cells,preferably into eukaryotic cells, such as into mammalian cells. Suchmethods encompass, for example, electroporation, lipofection, e.g. basedon cationic lipids and/or liposomes, calcium phosphate precipitation,nanoparticle based transfection, virus based transfection, ortransfection based on cationic polymers, such as DEAE-dextran orpolyethylenimine etc. Preferably, the introduction is non-viral.

Vaccine: A vaccine is typically understood to be a prophylactic ortherapeutic material providing at least one antigen, preferably animmunogen. The antigen or immunogen may be derived from any materialthat is suitable for vaccination. For example, the antigen or immunogenmay be derived from a pathogen, such as from bacteria or virus particlesetc., or from a tumor or cancerous tissue. The antigen or immunogenstimulates the body's adaptive immune system to provide an adaptiveimmune response.

Vector: The term “vector” refers to a nucleic acid molecule, preferablyto an artificial nucleic acid molecule. A vector in the context of thepresent invention is suitable for incorporating or harboring a desirednucleic acid sequence, such as a nucleic acid sequence comprising acoding region. Such vectors may be storage vectors, expression vectors,cloning vectors, transfer vectors etc. A storage vector is a vectorwhich allows the convenient storage of a nucleic acid molecule, forexample, of an mRNA molecule. Thus, the vector may comprise a sequencecorresponding, e.g., to a desired mRNA sequence or a part thereof, suchas a sequence corresponding to the coding region and the 3′-UTR and/orthe 5′-UTR of an mRNA. An expression vector may be used for productionof expression products such as RNA, e.g. mRNA, or peptides, polypeptidesor proteins. For example, an expression vector may comprise sequencesneeded for transcription of a sequence stretch of the vector, such as apromoter sequence, e.g. an RNA polymerase promoter sequence. A cloningvector is typically a vector that contains a cloning site, which may beused to incorporate nucleic acid sequences into the vector. A cloningvector may be, e.g., a plasmid vector or a bacteriophage vector. Atransfer vector may be a vector which is suitable for transferringnucleic acid molecules into cells or organisms, for example, viralvectors. A vector in the context of the present invention may be, e.g.,an RNA vector or a DNA vector. Preferably, a vector is a DNA molecule.Preferably, a vector in the sense of the present application comprises acloning site, a selection marker, such as an antibiotic resistancefactor, and a sequence suitable for multiplication of the vector, suchas an origin of replication. Preferably, a vector in the context of thepresent application is a plasmid vector.

Vehicle: A vehicle is typically understood to be a material that issuitable for storing, transporting, and/or administering a compound,such as a pharmaceutically active compound. For example, it may be aphysiologically acceptable liquid which is suitable for storing,transporting, and/or administering a pharmaceutically active compound.

Variant of a sequence: A variant of a nucleic acid sequence or an aminoacid sequence typically differs from the original sequence in one ormore residues, such as one or more substituted, inserted and/or deletednucleotides or amino acid residues. Preferably, these variants have thesame biological function or specific activity compared to thefull-length molecule. In the context of the present invention, a variantof a nucleic acid sequence or of an amino acid sequence is preferably atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%,even more preferably at least 80%, even more preferably at least 90%,even more preferably at least 95%, most preferably at least 99%,identical to a reference sequence. It is further preferred that a“variant” as used herein comprises or consists of a continuous stretchof entities, such as nucleotides or amino acid residues, correspondingto a continuous stretch of entities in the reference molecule, whichrepresents at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably atleast 70%, more preferably at least 80%, even more preferably at least90%, even more preferably at least 95%, most preferably at least 99%, ofthe total (i.e. full-length) reference molecule.

Method for Modifying the Retention Time of an RNA on a ChromatographicColumn:

One aspect of the present invention concerns a method for modifying theretention time of RNA on a chromatographic column. In particular, theinvention provides a method for purifying RNA, such as chromatographicpurification of RNA from a mixture of RNA species. By modifying theretention time of RNA, the inventive method preferably allows forphysical separation of said RNA.

According to preferred embodiments, the invention relates to a methodfor modifying the retention time of an RNA on a chromatographic column,wherein the method comprises a step of adapting the RNA (sequence) byaltering the number of adenine (A) and/or uracil (U) nucleotides in theRNA sequence with respect to the number of A and/or U nucleotides in theoriginal RNA sequence.

The inventors surprisingly found that the total number of A and Unucleotides in an RNA influences the retention time of an RNA on achromatographic column in a chromatographic process, such as highpressure (high pressure) liquid chromatography (HPLC), more preferablyreversed-phase HPLC.

As used herein, the term ‘retention time’ typically refers to the amountof time a compound, such as RNA, spends on a chromatographic column,after it has been injected. If two compounds in a sample, e.g. two RNAspecies in an RNA mixture, have different retention times, each compoundwill spend a different amount of time on the chromatographic column.Retention times are usually quoted in units of seconds or minutes.

In this context, it was also found by the inventors that the separationfactor of a given chromatographic method can be modulated by alteringthe number of adenine (A) and/or uracil (U) nucleotides in an RNAsequence. An increased difference between at least two RNA species inthe number of A and/or U nucleotides was found to be correlated with anincreased separation factor (cf. FIG. 16). The inventive method maytherefore be employed for increasing the separation factor of achromatographic method by modifying the number of adenine (A) and/oruracil (U) nucleotides in an RNA sequence. A decreased differencebetween at least two RNA species in the number of A and/or U nucleotidesis correlated with a decreased separation factor. The inventive methodmay therefore be employed for decreasing the separation factor of achromatographic method by modifying the number of adenine (A) and/oruracil (U) nucleotides in an RNA sequence.

As used herein, the term “RNA species” typically relates to a pluralityof RNA molecules or (at least one RNA molecule) having the samenucleotide sequence.

Preferably, the retention time of an RNA species is the time requiredfor said RNA species to migrate, or elute, from the column, measuredfrom the instant the sample is injected into the mobile phase stream tothe point at which the peak maximum occurs. Alternatively, the retentiontime may also be determined by measuring the time from the instant thesample is injected to the appearance of the first unretained RNA at theoutlet. In preferred embodiments, the dependence of the retention timeon a given flow rate is removed by calculating the correspondingretention volume, which is typically calculated as the retention timemultiplied by the volumetric flow rate of the mobile phase. In preferredembodiments, the retention volume may thus be modified by the methodaccording to the invention.

The retention time of RNA is modified by the inventive method, whereinthe retention time is preferably increased or decreased. In particular,the retention time of an original RNA (sequence) is modified by alteringthe number of A and/or U nucleotides in said original RNA (sequence),thereby obtaining an adapted RNA (sequence) having a modified retentiontime. In this context, the term ‘original RNA (sequence)’ may refer toany RNA (sequence), which retention time may be modified by the methodas described herein. Hence, an ‘original RNA (sequence)’ may be a wildtype RNA sequence. Furthermore, also a modified RNA sequence, preferablyan optimized RNA sequence, more preferably as described herein (e.g. anRNA sequence, which has been modified (optimized) with respect to itsG/C content), may be used as an ‘original RNA (sequence)’ in the meaningof the present invention. The term ‘adapted RNA (sequence)’ as usedherein preferably refers to an RNA (sequence), which is derived from theoriginal RNA (sequence), from which it differs by the total number of Aand/or U nucleotides. In other words, an adapted RNA (sequence) may beobtained by the inventive method, in particular by altering the numberof A and/or U nucleotides in an original RNA (sequence) and thuspreferably modifying the RNA's retention time. In a preferredembodiment, an adapted RNA (sequence) is obtained by the inventivemethod, which differs from the original RNA (sequence) only in thenumber of A and/or U nucleotides or in the corresponding sequencechanges at the respective nucleotide positions.

The retention time of an original RNA is thus typically modified by theinventive method, wherein the retention time of the adapted RNA ispreferably modified, more preferably increased or decreased, withrespect to the retention time of the original RNA.

As used herein, the term ‘RNA (sequence)’ may refer to an RNA as anindividual compound or to an RNA species, preferably as describedherein, or to the nucleotide sequence of a given RNA or RNA species. Inthe context of the present invention, the term ‘RNA species’ typicallyrelates to an RNA defined by a certain nucleotide sequence. A pluralityof RNA molecules having the same nucleotide sequence may also bereferred to herein as ‘RNA species’.

In particular, the term ‘RNA species’ as used herein may refer to afirst RNA species, such as a first plurality of RNA molecules sharingthe same nucleotide sequence (or to a population of RNA moleculessharing the same nucleotide sequence), wherein said RNA species ispreferably present in a mixture with a second (or further) RNA species,such as with a second (or further) plurality of RNA molecules sharing anucleotide sequence, which is distinct from the nucleotide sequence ofthe first RNA species (or with a second (or further) population of RNAmolecules sharing a nucleotide sequence, which is distinct from thenucleotide sequence of the first RNA species).

The term ‘RNA’ as used herein may refer to any type of RNA withoutlimitation. Preferably, the term RNA refers to a molecule or to amolecule species selected from the group consisting of long-chain RNA,coding RNA, non-coding RNA, single stranded RNA (ssRNA), double strandedRNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA(mRNA), RNA oligonucleotides, small interfering RNA (siRNA), smallhairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs,riboswitches, immunostimulating RNA (isRNA), ribozymes, aptamers,ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviralRNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA(snoRNA), microRNA (miRNA), circular RNA (circRNA), and aPiwi-interacting RNA (piRNA). Preferably, the RNA is a coding RNA.According to a particularly preferred embodiment, the method is used formodifying the retention time of a coding RNA, preferably an mRNA, morepreferably as described herein. According to preferred embodiments, thecoding RNA, preferably the mRNA, is a long-chain RNA as describedherein.

The method according to the invention is preferably used for modifyingthe retention time of a single-stranded RNA (ssRNA) or a double-strandedRNA (dsRNA), more preferably of an ssRNA. As used herein, the term‘single-stranded RNA’ or ‘ssRNA’ typically refers to an RNA moleculeconsisting of a single RNA strand, which may optionally comprise (e.g.based on the conditions), secondary structure elements (e.g. hairpins,stem-loops, etc.). In the context of the present invention, the term‘double-stranded RNA’ or ‘dsRNA’ typically refers to an RNA moleculecomprising two RNA strands, which preferably form a (hetero)duplex.

Preferably, RNA as used herein comprises more than 30 nucleotides. Morepreferably, the RNA is not selected from siRNA, small hairpin RNA,microRNA or small nuclear RNA (snRNA). In a particularly preferredembodiment, the RNA as used herein is not siRNA.

According to a preferred embodiment, the method is used for modifyingthe retention time of a long-chain RNA. The term ‘long-chain RNA’ asused herein herein typically refers to an RNA molecule, preferably asdescribed herein, which preferably comprises at least 30 nucleotides.Alternatively, a long-chain RNA may comprise at least 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750 or at least 800 nucleotides. Along-chain RNA molecule may further comprise at least 100 nucleotides,even more preferably at least 200 nucleotides. A long-chain RNA, in thecontext of the present invention, further preferably comprises from 30to 50.000 nucleotides, from 30 to 20000 nucleotides, from 100 to 20000nucleotides, from 200 to 20000 nucleotides, from 200 to 15000nucleotides, from 500 to 20000 nucleotides, from 800 to 20000nucleotides, from 800 to 5000 nucleotides or from 800 to 2000nucleotides. The term ‘long-chain RNA’ as used herein is not limited toa certain type of RNA, but merely refers to the number of nucleotidescomprised in said RNA. For example, the long-chain RNA may be a codingRNA, preferably an mRNA, more preferably as described herein.

According to preferred embodiments of the invention, the number of Aand/or U nucleotides in the original RNA sequence is determined. In thecontext of the present invention, the phrase ‘number of A and/or Unucleotides’ may refer to the total number of adenine (A) nucleotides,to the total number of uracil (U) nucleotides or, more preferably, tothe total number of A and U nucleotides. The number of A and/or Unucleotides is preferably increased or decreased to a target number,which may be a known number or a number, which has been pre-determined,preferably as described herein. That target number is preferablyselected according to the respective purpose. The modification of theretention time is typically the greater the larger the difference isbetween the target number and the original number of A and/or Unucleotides in the original RNA sequence.

Accordingly, the inventive method comprises a step of determining thenumber of A and/or U nucleotides in the original RNA sequence and a stepof increasing or decreasing said number of A and/or U nucleotides inorder to obtain the adapted RNA sequence.

As used herein, the term ‘adenine nucleotide’ or ‘A nucleotide’typically refers to an adenine nucleotide or to an analogue thereof,such as a chemically modified A nucleotide, preferably as describedherein. The term ‘uracil nucleotide’ or ‘U nucleotide’ may refer to anuracil nucleotide or to an analogue thereof, such as a chemicallymodified U nucleotide, preferably as described herein. In this context,the phrase ‘number of A and/or U nucleotides’ may also refer to thetotal number of adenine (A) nucleotides and adenine nucleotideanalogues, to the total number of uracil (U) nucleotides and uracilnucleotide analogues, or, more preferably, to the total number of Anucleotides, adenine nucleotide analogues, U nucleotides and Unucleotide analogues. In a similar manner, the terms ‘guaninenucleotide’ or ‘G nucleotide’ may refer to a guanine nucleotide or to ananalogue thereof, such as a chemically modified G nucleotide, preferablyas described herein. The term ‘cytosine nucleotide’ or ‘C nucleotide’may refer to a cytosine nucleotide or to an analogue thereof, such as achemically modified C nucleotide, preferably as described herein.

Wherever reference is made herein to an A, U, G, or C nucleotide, therespective nucleotide analogues, preferably as described herein, morepreferably a chemically modified nucleotide, is also comprised by thatterm.

Where reference is made herein to an ‘uracil nucleotide’, an ‘Unucleotide’ or an analogue of an uracil nucleotide, it is furtherunderstood that these terms may also relate to a thymine nucleotide inthe DNA sequence corresponding to an RNA sequence (e.g. in a DNA vectorencoding an RNA as described herein).

The length of the original RNA is preferably not modified so that thetotal number of nucleotides in the original RNA and the adapted RNA isessentially the same. Alternatively, the length of the original RNA mayalso be increased or decreased, e.g. by addition or deletion ofnucleotides.

In preferred embodiments, G and/or C nucleotides in the original RNAsequence are replaced with A and/or U nucleotides thereby altering,particularly increasing the number of A and/or U nucleotides in the RNAsequence with respect to the number of A and/or U nucleotides in theoriginal RNA sequence.

According to some embodiments, the inventive method comprises a step ofreplacing G and/or C nucleotides in the original RNA sequence with Aand/or U nucleotides.

Alternatively, A and/or U nucleotides in the original RNA sequence maybe replaced with G and/or C nucleotides thereby altering, particularlydecreasing the number of A and/or U nucleotides in the RNA sequence withrespect to the number of A and/or U nucleotides in the original RNAsequence.

According to some embodiments, the inventive method comprises a step ofreplacing A and/or U nucleotides in the original RNA sequence with Gand/or C nucleotides.

According to some embodiments, the method may also comprise a step ofintroducing additional nucleotides, preferably G and/or C nucleotides,more preferably A and/or U nucleotides, into an RNA sequence. Therein,nucleotides are preferably inserted into a non-coding region of theoriginal RNA sequence.

In preferred embodiments, the invention concerns a method for modifyingthe retention time of an RNA comprising at least one coding region. Inthese embodiments, the number of A and/or U nucleotides is preferablyaltered within a coding region, preferably as described herein. Forexample, G and/or C nucleotides in a coding region are replaced with Aand/or U nucleotides in order to increase the number of A and/or Unucleotides in the coding region. Alternatively, A and/or U nucleotidesin a coding region may be replaced with G and/or C nucleotides in orderto decrease the number of A and/or U nucleotides in the coding region.

In a particularly preferred embodiment, the number of A and/or Unucleotides in at least one coding region in an RNA is altered withoutmodifying the amino acid sequence encoded by the coding sequence of theoriginal RNA (sequence). The degeneracy of the genetic code allows forexchanges of codons in the coding region, which do not alter the encodedamino acid sequence. The method according to the invention thuspreferably comprises exchanging at least one codon in a coding region ofthe RNA with an alternative codon encoding the same amino acid, whereinthe alternative codon is preferably characterized by a number of Aand/or U nucleotides that is different from the number of A and/or Unumbers in the original codon. Reference is made to the descriptionherein of the inventive method for providing an adapted RNA sequence,where the selection of suitable codon exchanges is explained in moredetail.

The number of A and/or U nucleotides is preferably increased ordecreased to such an extent that the retention time is sufficientlymodified for a given purpose. Depending on the purpose, the number of Aand/or U nucleotides may be adapted to a pre-determined target number.For example, the number of A and/or U nucleotides in a given (original)RNA sequence may be adapted to the number or essentially the same numberof A and/or U nucleotides in the sequence of another RNA species, whichis also present in a mixture and which is to be co-purified with the RNAto be adapted. RNA species having essentially the same number of Aand/or U nucleotides are typically characterized by essentially the sameretention time on a chromatographic column, such as an HPLC column.According these RNA species elute from the column in the same fraction(peak). Alternatively, the target number may be selected such that thedifference from the number of A and/or U nucleotides in another RNAspecies in a mixture is sufficient for the adapted RNA to elute in aseparate fraction (peak) (which allows for determining the integrity ofeach RNA species).

In preferred embodiments, the number of A and/or U nucleotides in an RNAsequence is altered so that the number of A and/or U nucleotides in theadapted RNA sequence differs by at least 1, preferably by at least 2,more preferably by at least 3, even more preferably by at least 4, evenmore preferably by at least 5, even more preferably by at least 10, mostpreferably by at least 15, from the number of A and/or U nucleotides inthe original RNA sequence. Alternatively, the number of A and/or Unucleotides in the adapted RNA sequence may differ by at least 5,preferably by at least 10, more preferably by at least 20, even morepreferably by at least 30, even more preferably by at least 40, evenmore preferably by at least 50, most preferably by at least 60, from thenumber of A and/or U nucleotides in the original RNA sequence.Alternatively, the number of A and/or U nucleotides in the adapted RNAsequence may differ by at least 60, preferably by at least 80, morepreferably by at least 100, even more preferably by at least 120, evenmore preferably by at least 140, even more preferably by at least 160,most preferably by at least 200, from the number of A and/or Unucleotides in the original RNA sequence.

According to preferred embodiments, the method is used for increasingthe retention time of the adapted RNA with respect to the retention timeof the original RNA, wherein the number of A and/or U nucleotides in theadapted RNA sequence is increased by at least 1, preferably by at least2, more preferably by at least 3, even more preferably by at least 4,even more preferably by at least 5, even more preferably by at least 10,most preferably by at least 15, with respect to the number of A and/or Unucleotides in the original RNA sequence. Alternatively, the number of Aand/or U nucleotides in the adapted RNA sequence may be increased by atleast 5, preferably by at least 10, more preferably by at least 20, evenmore preferably by at least 30, even more preferably by at least 40,even more preferably by at least 50, most preferably by at least 60,with respect to the number of A and/or U nucleotides in the original RNAsequence. Alternatively, the number of A and/or U nucleotides in theadapted RNA sequence may be increased by at least 60, preferably by atleast 80, more preferably by at least 100, even more preferably by atleast 120, even more preferably by at least 140, even more preferably byat least 160, most preferably by at least 200, with respect to thenumber of A and/or U nucleotides in the original RNA sequence.

The method may also be used for decreasing the retention time of theadapted RNA with respect to the retention time of the original RNA,wherein the number of A and/or U nucleotides in the adapted RNA sequenceis decreased by at least 1, preferably by at least 2, more preferably byat least 3, even more preferably by at least 4, even more preferably byat least 5, even more preferably by at least 10, most preferably by atleast 15, with respect to the number of A and/or U nucleotides in theoriginal RNA sequence. Alternatively, the number of A and/or Unucleotides in the adapted RNA sequence may be decreased by at least 5,preferably by at least 10, more preferably by at least 20, even morepreferably by at least 30, even more preferably by at least 40, evenmore preferably by at least 50, most preferably by at least 60, withrespect to the number of A and/or U nucleotides in the original RNAsequence. Alternatively, the number of A and/or U nucleotides in theadapted RNA sequence may be decreased by at least 60, preferably by atleast 80, more preferably by at least 100, even more preferably by atleast 120, even more preferably by at least 140, even more preferably byat least 160, most preferably by at least 200, with respect to thenumber of A and/or U nucleotides in the original RNA sequence.

In some embodiments, the method comprises a step of adapting theoriginal RNA sequence by altering the number of A and/or U nucleotidesin such a manner that the ratio of the number of A nucleotides to thenumber of U nucleotides is from 0.2 to 5, preferably from 0.5 to 3, morepreferably from 1 to 3, even more preferably from 1 to 2.5, even morepreferably from 1.2 to 2, even more preferably from 1.4 to 2, even morepreferably from 1.5 to 2, most preferably from 1.6 to 2.

According to some embodiments, the retention time of RNA on achromatographic column is modified, wherein altering the number of Anucleotides in the RNA sequence has a stronger impact on the retentiontime of the RNA than altering the number of U nucleotides of the RNAsequence. Preferably, the change in retention time achieved by alteringthe number of A nucleotides is about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9 or 2 times, preferably 1.5 times, stronger than the change inretention time achieved by altering the same number of U nucleotides.

In some embodiments, the ratio of the number of A nucleotides to thenumber of U nucleotides in the RNA sequence is increased (withoutchanging the total number of A and U nucleotides) with respect to theratio of the number of A nucleotides to the number of U nucleotides inthe original RNA sequence, thereby increasing the retention time of theadapted RNA.

Alternatively, the ratio of the number of A nucleotides to the number ofU nucleotides in the RNA sequence is decreased (without changing thetotal number of A and U nucleotides) with respect to the ratio of thenumber of A nucleotides to the number of U nucleotides in the originalRNA sequence, thereby decreasing the retention time of the adapted RNA.

Each embodiment of the inventive method, which comprises a step ofaltering the number of A and/or U nucleotides in the RNA sequence in anymanner that leads to a desired modification of the retention time(decrease or increase) may be used as individual step or in anycombination thereof.

Accordingly, as defined herein, an increase of the retention time of theRNA may preferably be obtained by replacing G and/or C nucleotides inthe original RNA sequence, preferably in the coding region, with Aand/or U nucleotides and/or by introducing additional A and/or Unucleotides into the original RNA sequence, preferably into an UTRregion, and/or by increasing the ratio of the number of A nucleotides tothe number of U nucleotides (without changing the total number of A andU nucleotides) in the RNA sequence with respect to the ratio of thenumber of A nucleotides to the number of U nucleotides in the originalRNA sequence, wherein all of these adaptations are suitably appliedwithout modifying the amino acid sequence encoded by the coding regionof the original RNA (sequence).

Accordingly, as defined herein, a decrease of the retention time of theRNA may be obtained by replacing A and/or U nucleotides in the originalRNA sequence, preferably in the coding region, with G and/or Cnucleotides and/or by introducing additional G and/or C nucleotides intothe original RNA sequence, preferably into an UTR region, and/or bydecreasing the ratio of the number of A nucleotides to the number of Unucleotides (without changing the total number of A and U nucleotides)in the RNA sequence with respect to the ratio of the number of Anucleotides to the number of U nucleotides in the original RNA sequence,wherein all of these adaptations are suitably applied without modifyingthe amino acid sequence encoded by the coding region of the original RNA(sequence).

In preferred embodiments, the method comprises a step of adapting theoriginal RNA sequence by altering the number of A and/or U nucleotidesin the RNA sequence as described herein with respect to a method forpurifying at least one RNA species from a mixture and/or as describedherein with respect to a method for co-purifying at least two RNAspecies from a mixture.

The method according to the invention is preferably provided as a methodfor modifying the retention time of RNA on a chromatographic column,wherein the chromatographic column is an HPLC column. In this context,the method is preferably used for modifying the retention time of RNAunder conditions suitable for analytical or preparative purification ofRNA. Chromatographic procedures and experimental conditions suitable forpurifying RNA on an analytical or on a preparative scale, in particularby RP-HPLC, are known in the art and were described, for example, in WO2008/077592, which is hereby incorporated by reference in its entirety.

In preferred embodiments, the method is used for modifying the retentiontime of RNA on a chromatographic column in a reversed-phase HPLC(RP-HPLC). Preferably, the RP-HPLC is as described in WO 2008/077592.According to a preferred embodiment, the chromatographic columncomprises a reversed phase as a stationary phase. In reversed-phasechromatography, a nonpolar compound is preferably used as the stationaryphase and a polar solvent, such as an aqueous solution of e.g.acetonitrile and/or methanol, is used as the mobile phase for elution.

The column, which is used as stationary phase, may be provided in beadform or as a monolithic column, which is preferably a polymerized‘block’, i.e. a block which fills a substantial part of thechromatography column. Irrespective of its precise nature, the polymericstationary phase, in particular if used for preparative purification, ispreferably porous in its nature, which means that the beads or the blockare characterized by pores. In other embodiments, the reversed phase isa non-porous reversed phase.

In a preferred embodiment, in particular if used for preparativepurification, a porous reversed phase material is provided with aparticle size of 8.0 μm to 50 μm, in particular 8.0 to 30, still morepreferably 8.0 to 25 μm. The reversed phase material may be present inthe form of small beads. The method according to the invention may beperformed particularly favourably with a porous reversed phase with thisparticle size, optionally in bead form, wherein particularly goodseparation results are obtained for preparative purification.

In a preferred embodiment, the reversed phase has a pore size of 1000 Åto 5000 Å, in particular a pore size of 1000 Å to 4000 Å, morepreferably 1500 Å to 4000 Å, 2000 Å to 4000 Å or 2500 Å to 4000 Å.Particularly preferred pore sizes for the reversed phases are 1000 Å to2000 Å, more preferably 1000 Å to 1500 Å and most preferably 1000 Å to1200 Å or 3500-4500 Å. In other embodiments, the reversed phase is aporous reversed phase with undefined pore size.

A pore size of 1000 Å to 5000 Å, in particular a pore size of 1000 Å to4000 Å, more preferably 1500 Å to 4000 Å, 2000 Å to 4000 Å or 2500 Å to4000 Å may be suitable to separate an RNA from other components of amixture, the RNA preferably having a size as mentioned herein withrespect to the long-chain RNA, e.g. of up to about 15000 nucleotides (assingle stranded RNA molecule) or base pairs (as double stranded RNAmolecule), in particular 100 to 10000, more preferably 500 to 10000nucleotides or base pairs, even more preferably 800 to 5000 nucleotidesor base pairs and even more preferably 800 to 2000 nucleotides or basepairs. However, the pore size of the reversed phase may also be selectedin dependence of the size of the RNA to be separated, i.e. a larger poresize may be selected, if larger RNA molecules are to be separated andsmaller pore sizes may be selected, if smaller RNA molecules may beselected. This is due to the effect, that the retention of the RNAmolecules and the separation not only depends on the interaction of the(reversed) phase but also on the possibility of molecules to get insidethe pores of the matrix and thus provide a further retention effect.Without being limited thereto, e.g. a pore size for the reversed phaseof about 2000 Å to about 5000 Å, more preferably of about 2500 to about4000, most preferably of about 3500 to about 4500 Å, may thus be used toseparate larger RNA molecules, e.g. RNA molecules of 100 to 10000, morepreferably 500 to 10000 nucleotides or base pairs, even more preferably800 to 5000 nucleotides or base pairs and even more preferably 800 to2000 nucleotides or base pairs. Alternatively, without being limitedthereto, a pore size of for the reversed phases of about 1000 Å to about2500 Å, more preferably of about 1000 Å to about 2000 Å, and mostpreferably of about 1000 Å to 1200 Å may be used to separate smaller RNAmolecules, e.g. RNA molecules of about 30-1000, 50-1000 or 100-1000 or20-200, 20-100, 20-50 or 20-30 nucleotides may also be separated in thisway.

In general, any material known to be used as reverse phase stationaryphase, in particular any polymeric material may be used forchromatographic method in the context of the present invention. Thestationary phase may be composed of organic and/or inorganic material.Examples for polymers to be used for the present invention are(non-alkylated or alkylated) polystyrenes, (non-alkylated or alkylated)polystyrenedivinylbenzenes, silica gel, silica gel modified withnon-polar residues, particularly silica gel modified with alkylcontaining residues, more preferably with butyl-, octyl and/or octadecylcontaining residues, silica gel modified with phenylic residues,polymethacrylates, etc. or other materials suitable e.g. for gelchromatography or other chromatographic methods as mentioned above, suchas dextran, including e.g. Sephadex® and Sephacryl® materials, agarose,dextran/agarose mixtures, polyacrylamide, etc.

Preferably, the chromatographic column comprises a material selectedfrom the group consisting of polystyrene, a non-alkylated polystyrene,an alkylated polystyrene, a polystyrenedivinylbenzene, a non-alkylatedpolystyrenedivinylbenzene, an alkylated polystyrenedivinylbenzene, asilica gel, a silica gel modified with non-polar residues, a silica gelmodified with alkyl containing residues, selected from butyl-, octyland/or octadecyl containing residues, a silica gel modified withphenylic residues, or a polymethacrylate.

In a particularly preferred embodiment, the chromatographic columncomprises a material selected from the group consisting of a polystyrenepolymer, a non-alkylated polystyrene polymer, an alkylated polystyrenepolymer, a non-alkylated polystyrenedivinylbenzene polymer, an alkylatedpolystyrenedivinylbenzene, a silica gel, a silica gel modified withnon-polar residues, particularly silica gel modified with alkylcontaining residues, more preferably with butyl-, octyl and/or octadecylcontaining residues, porous silica gel modified with phenylic residues,polymethacrylates. All these materials may be porous, preferably asdescribed herein, or non-porous.

Stationary phases with polystyrenedivinylbenzene are known per se. Theper se known polystyrenedivinyl-benzenes already used for HPLC methods,which are commercially obtainable, may be used for the chromatographicmethod in the context of the invention.

In preferred embodiments, a non-alkylated porouspolystyrenedivinylbenzene is particularly preferred, which, withoutbeing limited thereto, may have in particular a particle size of 8.0±1.5μm, in particular 8.0±0.5 μm, and a pore size of 1000-1500 Å, inparticular 1000-1200 Å or 3500-4500 Å.

In further embodiments, an alkylated, macro porous monolithicpolystyrenedivinylbenzene is particularly preferred, which, withoutbeing limited thereto, may have a pore size distribution of about 0.1um-10 μm, particularly about of 1 um-10 μm, more particularly of about 1um-6 μm.

In further embodiments a porous silica gel is used. The porous silicagel may be prepared from tetraethoxysilane and bis(triethoxysilyl)ethaneused in a 4:1 molar ratio and the porous silica gel is modified with anoctadecyl carbon chain. Such porous silica gel is commerciallyavailable, e.g. XBRIDGE™ OST C18 from Waters or AQUITY UPLC OST C18 fromWaters.

The silica gel may have a particle size of 0.5 to 5 μm, preferably of0.7 to 4 μm, more preferably of 1 to 3 μm, even more preferably of 1.5to 2 μm and most preferably of 1.7 μm. The pore size of the poroussilica gel may be 50 to 300 Å, preferably 70 to 250 Å, more preferably100 to 200 Å, even more preferably 120 to 170 Å and most preferably itis 135 Å.

In a preferred embodiment, a mixture of an aqueous solvent and anorganic solvent is used in HPLC as the mobile phase for eluting the RNA.It is favourable for a buffer to be used as the aqueous solvent whichhas in particular a pH of 6.0-8.0, for example of about 7, for example7.0. Preferably the buffer is triethylammonium acetate, more preferablya 0.02 M to 0.5 M, in particular 0.08 M to 0.12 M, even more preferablyan about 0.1 M triethylammonium acetate buffer. In a preferredembodiment, the organic solvent which is used in the mobile phase isacetonitrile, methanol, ethanol, 1-propanol, 2-propanol and acetone or amixture thereof, very particularly preferably acetonitrile. In aparticularly preferred embodiment, the mobile phase is a mixture of 0.1M triethylammonium acetate, pH 7, and acetonitrile.

Any one of the individual steps or features (or a combination of suchsteps or features) described herein with respect to the method formodifying the retention time of an RNA may also be applied with respectto any one of the other aspects of the present invention as describedherein, in particular to the method for purifying at least one RNAspecies from a mixture of at least two RNA species, the method forco-purifying at least two RNA species from a mixture of at least two RNAspecies, the method for harmonizing the numbers of A and/or Unucleotides in the sequences of at least two RNA species, or the methodfor providing an adapted RNA as described herein.

Method for Purifying at Least One RNA Species from a Mixture:

In a further aspect, the present invention concerns a method forpurifying at least one RNA species from a mixture of at least two RNAspecies. This method is preferably characterized by any one of themethod steps or any one of the features (or a combination thereof)described herein with regard to other aspects of the invention, inparticular with regard to the method for modifying the retention time ofan RNA or the method for co-purifying at least two RNA species from amixture comprising at least two RNA species.

In preferred embodiments, the method for purifying at least one RNAspecies from a mixture of at least two RNA species comprises:

a) a step of adapting the sequence of the at least one RNA species byaltering the number of A and/or U nucleotides with respect to the numberof A and/or U nucleotides in the original sequence; andb) a chromatographic step, wherein the adapted RNA species is separatedfrom at least one other RNA species.

In the context of the present invention, the term ‘purifying’ isunderstood to mean that a desired RNA (species) in a sample is separatedand/or isolated from another compound, which is further present in amixture, typically a solution. These other compounds may be, forexample, impurities or other RNA species, from which the desired RNA(species) is to be separated. Thus, after HPLC purification the RNA ispresent in a purer form than in the originally introduced RNA-containingsample prior to HPLC purification. Undesired constituents ofRNA-containing samples which therefore need to be separated may inparticular be impurities derived from an in vitro transcriptionreaction, such as degraded RNA fragments or fragments, which have arisenas a result of premature termination of transcription, or alsoexcessively long transcripts if plasmids are not completely linearized.In addition, impurities such as enzymes, for example RNases andpolymerases, and nucleotides may be separated.

Using the methods according to the invention, RNA is purified which hasa higher purity after purification than the starting material. It isdesirable in this respect for the degree of purity to be as close aspossible to 100%. A degree of purity of more than 70%, in particular80%, very particularly 90% and most favourably 99% or more may beachieved in this way.

According to a preferred embodiment, the method for purifying at leastone RNA species from a mixture of at least two RNA species comprises instep b) an HPLC process, preferably an RP-HPLC process, as describedherein and, in further detail, in WO 2008/077592.

According to preferred embodiments, the method for purifying at leastone RNA species from a mixture of at least two RNA species is used as ananalytical purification process, e.g. during quality control in RNAmanufacture. Preferably, the method is used for determining the identityand/or integrity of at least one RNA species. For example, the sequenceof one RNA, which may have the same or essentially the same retentiontime as another RNA species in the mixture and thus elutes in onefraction (peak) together with that other RNA species, may be adapted inorder for the adapted RNA species to have a distinct retention time,which results in separation of the fractions (peaks), into which therespective RNA species elute (see also FIG. 1E).

According to a preferred embodiment, the method for purifying at leastone RNA species from a mixture of at least two RNA species furthercomprises a step of determining the identity and/or integrity of atleast one RNA species.

The term “integrity” describes whether complete RNA molecules arepresent in a sample applied to the analytical purification process asdescribed herein. If complete RNA molecules are present, the RNAmolecule of the at least one RNA species in a mixture of at least twoRNA species will have the expected length. If incomplete RNA moleculesare present, the length of the RNA molecule will be different from theexpected or known length. Low integrity or differences in the length ofthe RNA molecules could be due to, amongst others, degradation,cleavage, incorrect base pairing, lack of or incomplete capping, lack ofor incomplete polyadenylation, or incomplete transcription. This willbecome evident in a broadening of the HPLC peak of the RNA during theanalytical purification process as described herein.

In an analytical purification process, smaller amounts of RNA aretypically subjected to an HPLC. A typical amount of RNA subjected toanalytical purification process may be smaller than about 5 μg, e.g.about 4 μg or less, about 3 μg or less, about 2 μg or less, about 1 μgor less, about 500 ng or less, about 400 ng or less, about 300 ng orless, about 200 ng or less, about 100 ng or less, or about 50 ng orless. Preferably, the amount of RNA subjected to an analyticalpurification process is in a range selected from about 1 ng to about 5μg, from about 10 ng to about 4 μg, from about 10 ng to about 3 μg, fromabout 10 ng to about 2 μg, from about 10 ng to about 1 μg, or from about50 ng to 5 μg. More preferably, the amount of RNA subjected to ananalytical purification process is at least about 10 ng.

The method for purifying at least one RNA species from a mixture of atleast two RNA species may also be used on a preparative scale. Inparticular, the method according to the invention may be employed inorder to purify an adapted RNA sequence from a mixture, which has beenpre-purified in at least one other purification step (e.g. by HPLC).

In a preparative purification process, larger amounts of RNA aretypically subjected to an HPLC. A typical amount of RNA subjected to apreparative purification process may be larger than 5 μg, e.g. about 10μg or more, about 100 μg or more, about 1 mg or more, about 10 mg ormore, about 100 mg or more, about 1 g or more, about 10 g or more, about100 g or more, about 1 kg or more, about 10 kg or more. Preferably, theamount of RNA subjected to a preparative purification process is in arange selected from about 5 μg to about 10 kg, from about 5 μg to about1 kg, from about 5 μg to about 500 g, from about 5 μg to about 100 g,from about 5 μg to about 50 g, or from about 5 μg to about 10 g.

Preferably, step a) of the method comprises a step of modifying theretention time of at least one RNA species from a mixture of at leasttwo RNA species on a chromatographic column as described herein.

In certain embodiments, the retention time of the at least one RNAspecies to be purified (in its unmodified/non-adapted state,corresponding to the original RNA (sequence)) is essentially the same asthe retention time of the RNA species, from which it is to be separatedor the retention times are so close that the RNA species elute from achromatographic column in the same fraction (peak) or in overlappingfractions (peaks) that do not allow for physical separation of the RNAspecies, and in addition, do not allow for determining the integrity ofat least one RNA species.

In preferred embodiments, the number of A and/or U nucleotides in thesequence of the at least one RNA species to be purified is altered asdescribed herein so that physical separation of the at least one RNAspecies from other components in a mixture is feasible bychromatography, preferably by HPLC, more preferably by RP-HPLC. Theretention time of the at least one RNA species is preferably modifiedsuch that the RNA species elutes in a separate fraction (peak), whichdoes not comprise at least one other compound (e.g. another RNA speciesor impurities such as abortive sequences etc.), from which the at leastone RNA species is to be separated. The modified retention time of theat least one adapted RNA species is different from the retention time ofat least one further RNA species in the mixture, from which it maypreferably be physically separated, more preferably by HPLC.

According to a particularly preferred embodiment, step a) of the methodcomprises adapting the sequence of the at least one RNA species to bepurified so that the number of A and/or U nucleotides in the adaptedsequence of the at least one RNA species differs by at least 1,preferably by at least 2, more preferably by at least 3, even morepreferably by at least 4, even more preferably by at least 5, even morepreferably by at least 10, most preferably by at least 15, from thenumber of A and/or U nucleotides in the sequence of at least one otherRNA species, which is present in the mixture and from which the adaptedRNA species is to be separated. Alternatively, the number of A and/or Unucleotides in the adapted sequence of the at least one RNA species maydiffer by at least 5, preferably by at least 10, more preferably by atleast 20, even more preferably by at least 30, even more preferably byat least 40, even more preferably by at least 50, most preferably by atleast 60, from the number of A and/or U nucleotides in the sequence ofanother RNA species in the mixture.

In preferred embodiments, e.g. an analytical purification process, stepa) of the method comprises adapting the sequence of the at least one RNAspecies to be purified so that the number of A and/or U nucleotides inthe adapted sequence of the at least one RNA species may differ by atleast 10, preferably by at least 20, more preferably by at least 30,even more preferably by at least 40, even more preferably by at least50, even more preferably by at least 60, most preferably by at least 70,from the number of A and/or U nucleotides in the sequence of another RNAspecies in the mixture.

In preferred embodiments, e.g. a preparative purification process, stepa) of the method comprises adapting the sequence of the at least one RNAspecies to be purified so that the number of A and/or U nucleotides inthe adapted sequence of the at least one RNA species may differ by atleast 80, preferably by at least 100, more preferably by at least 120,even more preferably by at least 140, even more preferably by at least180, even more preferably by at least 200, most preferably by at least220, from the number of A and/or U nucleotides in the sequence ofanother RNA species in the mixture.

According to preferred embodiments, e.g. in an analytical purificationprocess, the sequence of the at least one RNA species to be purified isadapted so that the number of A and/or U nucleotides in the adapted RNAdiffers from the respective number in the sequence of at least one otherRNA species in the mixture, from which it is to be separated by anumber, which is equal to or greater than the total number ofnucleotides in the sequence of the at least one RNA to be purifiedmultiplied by a factor of at least 0.05, at least 0.06, at least 0.07,at least 0.08, at least 0.09, at least 0.1 or at least 0.15. In apreferred embodiment, the number of A and/or U nucleotides in theadapted RNA differs from the respective number in the sequence of the atleast one other RNA species in the mixture, from which it is to beseparated by a number, which is equal to or greater than the totalnumber of nucleotides in the sequence of the at least one RNA to bepurified multiplied by a factor of 0.065. In these embodiments, the atleast one RNA species to be purified preferably has the same length(i.e. comprises the same total amount of nucleotides) as the RNAspecies, from which it is to be separated. More preferably, the lengthsof the RNA species differ by not more than about 20%, not more thanabout 10%, not more than about 5%, not more than about 4%, not more thanabout 3%, not more than about 2%, or not more than about 1%.

According to preferred embodiments, e.g. in an analytical purificationprocess, the sequence of the at least one RNA species to be purified isadapted so that the number of A and/or U nucleotides in the adapted RNAdiffers from the respective number in the sequence of at least one otherRNA species in the mixture, from which it is to be separated by anumber, which is equal to or greater than the total number ofnucleotides in the sequence of the at least one RNA to be purifiedmultiplied by a factor of at least 0.01, at least 0.02, at least 0.03,at least 0.04, or at least 0.05. In these embodiments, the at least oneRNA species to be purified preferably has the same length (i.e.comprises the same total amount of nucleotides) as the RNA species, fromwhich it is to be separated. More preferably, the lengths of the RNAspecies differ by not more than about 20%, not more than about 10%, notmore than about 5%, not more than about 4%, not more than about 3%, notmore than about 2%, or not more than about 1%.

According to preferred embodiments, e.g. in a preparative purification,the sequence of the at least one RNA species to be purified is adaptedso that the number of A and/or U nucleotides in the adapted RNA differsfrom the respective number in the sequence of at least one other RNAspecies in the mixture, from which it is to be separated by a number,which is equal to or greater than the total number of nucleotides in thesequence of the at least one RNA to be purified multiplied by a factorof at least 0.20, at least 0.22, at least 0.24, at least 0.26, at least0.28, at least 0.30 or at least 0.35. In these embodiments, the at leastone RNA species to be purified preferably has the same length (i.e.comprises the same total amount of nucleotides) as the RNA species, fromwhich it is to be separated. More preferably, the lengths of the RNAspecies differ by not more than about 20%, not more than about 10%, notmore than about 5%, not more than about 4%, not more than about 3%, notmore than about 2%, or not more than about 1%.

Method for Co-Purifying at Least Two RNA Species from a Mixture:

According to a further aspect, the present invention provides a methodfor co-purifying at least two RNA species from a mixture comprising atleast two RNA species, wherein the method comprises:

a) a step of adapting the sequence of at least one RNA species byaltering the number of A and/or U nucleotides in the RNA with respect tothe number of A and/or U nucleotides in the original RNA sequence; andb) a chromatographic step, wherein the at least one RNA species havingan adapted sequence and at least one further RNA species areco-purified.

This method preferably comprises any one of the method steps or any oneof the features (or a combination thereof) described herein with regardto other aspects of the invention, in particular with regard to themethod for modifying the retention time of an RNA or with regard to themethod for purifying at least one RNA species from a mixture of at leasttwo RNA species.

The method is preferably used for co-purifying at least two RNA speciesfrom a mixture comprising at least two RNA species, wherein the at leasttwo RNA species (in their unmodified/non-adapted state) arecharacterized by distinct retention times, which result in elution ofthe at least two RNA species in separate fractions (peaks), which do notallow for co-purification.

In the method for co-purifying at least two RNA species from a mixturecomprising at least two RNA species, the number of A and/or Unucleotides in an original RNA sequence is preferably altered in such amanner that the retention time of the RNA is essentially identical to orat least sufficiently close to the retention time of another compound(such as another RNA species), which is further present in a mixturetogether with the adapted RNA, for the adapted RNA and the othercompound (e.g. another RNA species) to be co-purified by achromatographic procedure, preferably by HPLC, more preferably byRP-HPLC as described herein. The adapted RNA and the other compound(e.g. another RNA species) preferably elute from the chromatographiccolumn together in one fraction (or appear in the chromatogram as onesingle peak, respectively). The adaptation of the number of A and/or Unucleotides in an RNA (sequence) in order to obtain an adapted RNAhaving essentially the same retention time as another RNA species mayalso be referred to herein as ‘(peak) harmonization’ or ‘harmonizationof the retention times’.

More preferably, the number of A and/or U nucleotides in an original RNAsequence is preferably altered in such a manner that the retention timeof the RNA (species) differs by not more than about 20%, preferably notmore than about 10%, not more than about 5%, not more than about 4%, notmore than about 3%, not more than about 2%, not more than about 1%, notmore than 0.5% or not more than 0.1%, from the retention time of anothercompound (such as another RNA species), which is further present in amixture together with the adapted RNA.

In a further aspect, the present invention thus also provides a methodfor harmonizing the numbers of A and/or U nucleotides in the sequencesat least two RNA species, the method comprising adapting the sequence ofat least one RNA species by altering the number of A and/or Unucleotides in the RNA sequence with respect to the number of A and/or Unucleotides in the original RNA sequence. Said method preferably appliesany one of the steps or any one of the features (or a combinationthereof) described herein with respect to the method for co-purifying atleast two RNA species.

Hence, the retention times of harmonized RNA species as used hereinpreferably differ by not more than about 20%, more preferably by notmore than about 10%, by not more than about 5%, by not more than about4%, by not more than about 3%, by not more than about 2%, by not morethan about 1%, by not more than 0.5% or by not more than 0.1%. Even morepreferably, harmonized RNA species are characterized by essentiallyidentical retention times.

In a further aspect, the present invention provides a method forsynthesis of a mixture comprising at least two harmonized RNA species,preferably as described herein, the method comprising

a) a step comprising harmonizing the numbers of A and/or U nucleotidesin the sequences of at least two RNA species, wherein the sequence of atleast one RNA species is adapted by altering the number of A and/or Unucleotides in the RNA sequence with respect to the number of A and/or Unucleotides in the original RNA sequence; andb) a step of synthesis of the at least two harmonized RNA species.Step a) of this method preferably comprises adaptation of the RNAsequence of at least one of the RNA sequences by altering the number ofA and/or U nucleotides in the RNA sequence with respect to the number ofA and/or U nucleotides in the original RNA. In this respect and alsowith regard to step b), reference is made to the description of theinventive method for modifying the retention time of RNA on achromatographic column, the inventive method for purifying at least oneRNA from a mixture of at least two RNA species or the inventive methodfor co-purifying at least two RNA species from a mixture comprising atleast two RNA species. Any one of the features described herein withrespect to one or more of said methods preferably also apply to theinventive method for synthesis of a mixture comprising at least twoharmonized RNA species.

In some embodiments, step a) of the method for synthesis of a mixturecomprising at least two harmonized RNA species may comprise:

i) optionally, determining the total number of nucleotides in anoriginal RNA sequence;ii) determining the number of A and/or U nucleotides in the original RNAsequence;iii) determining the codons in the original RNA sequence that can bereplaced with at least one alternative codon without changing theencoded amino acid sequence;iv) adjusting the number of A and/or U nucleotides in the RNA sequenceto a pre-set number of A and/or U nucleotides by replacing at least oneoriginal codon with an alternative codon, wherein the alternative codonencodes the same amino acid as the original codon and is furthercharacterized in a higher content of A and/or U nucleotides.Step b) of the method for synthesis of a mixture comprising at least twoharmonized RNA species preferably comprises the separate synthesis ofthe at least two harmonized RNA species. In order to obtain a mixture ofharmonized RNA species, at least two harmonized RNA species arepreferably mixed together. Alternatively, step b) comprises synthesis ofthe at least two harmonized RNA species in one batch. The at least twoharmonized RNA species may be synthesized by any method known in theart, e.g. by chemical synthesis. Preferably, the harmonized RNA speciesare synthesized by RNA in vitro transcription.

The term “mixture” as used in this context preferably refers to a solid,semi-solid or liquid mixture. Preferably, the mixture comprising atleast two harmonized RNA species may be a liquid, more preferably anaqueous solution. Further suitable solvents, buffers and excipients areknown in the art and are also further described herein, in particularwith respect to the inventive composition. In preferred embodiments, themixture comprising at least two harmonized RNA species, which isobtained by the inventive method for synthesis of a mixture comprisingat least two harmonized RNA species, is a composition that is preferablycharacterized by any one of the features described with respect to thecomposition (comprising at least two RNA species) as described herein.

According to a preferred embodiment, the method for co-purifying atleast two RNA species (or the method for harmonizing the number of Aand/or U nucleotides in the sequences of at least two RNA species or themethod for synthesis of a mixture comprising at least two harmonized RNAspecies) comprises a step of determining a target number of A and/or Unucleotides, to which the number of A and/or U nucleotides in the atleast two RNA species to be co-purified (to be harmonized) is adapted.For example, if a mixture contains a multitude of RNA species, which arecharacterized by essentially the same retention time, and two or moreRNA species are to be separated from the remaining RNA species (i.e.co-purified), then a target number of A and/or U nucleotides isdetermined, preferably as described herein, which results in the RNAspecies to be co-purified (or harmonized) having essentially the sameretention time, thus eluting from a chromatographic column within thesame fraction (peak), allowing for (co-) separation of these RNA speciesfrom the undesired RNA species in the mixture.

In order for two (or more) RNA species to elute from a chromatographiccolumn, such as a (reversed-phase) HPLC column, within the same fraction(peak), thus allowing for co-purification of the two (or more) RNAspecies, preferably in an analytical scale purification, the numbers ofA and/or U nucleotides in the two (or more) RNA species preferablydiffer from each other by not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or 35. Inpreferred embodiments, the numbers of A and/or U nucleotides in the RNAspecies preferably differ from each other by not more than 2, morepreferably by not more than 3, even more preferably by not more than 4,even more preferably by not more than 5, most preferably by not morethan 6. In a further preferred embodiment, the numbers of A and/or Unucleotides in the RNA species differ from each other by not more than10, preferably by not more than 15, more preferably by not more than 20,even more preferably by not more than 25, most preferably by not morethan 30. It is particularly preferred, that the numbers of A and/or Unucleotides in the two or more RNA species are identical.

For preparative scale purification, in order for two (or more) RNAspecies to elute from a chromatographic column, such as a(reversed-phase) HPLC column, within the same fraction (peak), thusallowing for co-purification of the two (or more) RNA species, thenumbers of A and/or U nucleotides in the two (or more) RNA speciespreferably differ from each other by not more than 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150. In preferred embodiments, the numbers of A and/or Unucleotides in the RNA species preferably differ from each other by notmore than 2, more preferably by not more than 3, even more preferably bynot more than 4, even more preferably by not more than 5, mostpreferably by not more than 6. In a further preferred embodiment, thenumbers of A and/or U nucleotides in the RNA species differ from eachother by not more than 10, preferably by not more than 20, morepreferably by not more than 30, even more preferably by not more than40, most preferably by not more than 50. It is particularly preferred,that the numbers of A and/or U nucleotides in the two or more RNAspecies are identical.

According to a preferred embodiment, the numbers of A and/or Unucleotides in the RNA species, which are to be co-purified and whichpreferably have the same length, differ by a number, which is equal toor lower than the total number of nucleotides in the RNA multiplied by afactor equal to or lower than 0.008, equal to or lower than 0.0075,equal to or lower than 0.007, equal to or lower than 0.0065, equal to orlower than 0.006, equal to or lower than 0.0055, equal to or lower than0.005, equal to or lower than 0.0045, equal to or lower than 0.004,equal to or lower than 0.0035 or equal or lower than 0.003. Morepreferably, the numbers of A and/or U nucleotides in the RNA species,which are to be co-purified and which preferably have the same length,differ by a number, which is equal to or lower than the total number ofnucleotides in the RNA multiplied by a factor equal to or lower than0.0065, preferably equal to or lower than 0.0063.

According to preferred embodiments, the numbers of A and/or Unucleotides in the RNA species, which are to be co-purified and whichpreferably have the same length, differ by a number, which is equal toor lower than the total number of nucleotides in the RNA multiplied by afactor equal to or lower than 0.030, equal to or lower than 0.025, equalto or lower than 0.020, equal to or lower than 0.015, equal to or lowerthan 0.014, equal to or lower than 0.013, equal to or lower than 0.012,equal to or lower than 0.011, equal to or lower than 0.010, equal to orlower than 0.009 or equal or lower than 0.008.

Modified RNA:

According preferred embodiments of the invention, the RNA (species) asdescribed herein, may be in the form of modified RNA, wherein anymodification, as defined herein, may be introduced into the RNA. Amodification as defined herein preferably leads to an artificial RNA asdescribed herein, which is further stabilized.

According to one embodiment, the RNA, may thus be provided as a‘stabilized RNA’, preferably as a ‘stabilized mRNA’, that is to say asan RNA that is essentially resistant to in vivo degradation (e.g. by anexo- or endo-nuclease). Such stabilization can be effected, for example,by a modified phosphate backbone of an artificial RNA. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained in theRNA are chemically modified. Nucleotides that may be preferably used inthis connection contain e.g. a phosphorothioate-modified phosphatebackbone, preferably at least one of the phosphate oxygens contained inthe phosphate backbone being replaced by a sulfur atom. Stabilized RNAsmay further include, for example: non-ionic phosphate analogues, suchas, for example, alkyl and aryl phosphonates, in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group, orphosphodiesters and alkylphosphotriesters, in which the charged oxygenresidue is present in alkylated form. Such backbone modificationstypically include, without implying any limitation, modifications fromthe group consisting of methylphosphonates, phosphoramidates andphosphorothioates (e.g. cytidine-5′-O-(1-thiophosphate)).

In the following, specific modifications are described, each of which ispreferably capable of ‘stabilizing’ the RNA, preferably an mRNA, asdescribed herein.

Chemical Modifications:

The terms ‘nucleic acid modification’ or ‘RNA modification’ as usedherein may refer to chemical modifications comprising backbonemodifications as well as sugar modifications or base modifications.

In this context, a modified RNA as defined herein may contain nucleotideanalogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in the RNA asdefined herein are chemically modified. A sugar modification inconnection with the present invention is a chemical modification of thesugar of the nucleotides of the RNA as defined herein. Furthermore, abase modification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the artificialRNA. In this context, nucleotide analogues or modifications arepreferably selected from nucleotide analogues, which are applicable fortranscription and/or translation.

Sugar Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified RNA as described herein, can be modified in the sugar moiety.For example, the 2′ hydroxyl group (OH) can be modified or replaced witha number of different “wry” or “deoxy” substituents. Examples of“wry”-2′ hydroxyl group modifications include, but are not limited to,alkoxy or aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar); polyethyleneglycols (PEG),—O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon ofthe same ribose sugar; and amino groups (—O-amino, wherein the aminogroup, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl,arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylenediamine, polyamino) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and 0.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, an RNA can include nucleotides containing, forinstance, arabinose as the sugar.

Backbone Modifications:

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides, which may be incorporated into a modifiedRNA as described herein. The phosphate groups of the backbone can bemodified by replacing one or more of the oxygen atoms with a differentsubstituent. Further, the modified nucleosides and nucleotides caninclude the full replacement of an unmodified phosphate moiety with amodified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of the linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications:

The modified nucleosides and nucleotides, which may be incorporated intoa modified nucleic acid, preferably an mRNA as described herein canfurther be modified in the nucleobase moiety. Examples of nucleobasesfound in a nucleic acid such as RNA include, but are not limited to,adenine, guanine, cytosine and uracil. For example, the nucleosides andnucleotides described herein can be chemically modified on the majorgroove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyDadenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine,5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine or5′-O-(1-thiophosphate)-pseudouridine.

In further specific embodiments, a modified RNA may comprise nucleosidemodifications selected from 6-aza-cytidine, 2-thio-cytidine,alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine,8-azido-adenosine, 7-deaza-adenosine.

In some embodiments, the RNA as used herein comprises at least onecoding region as defined herein, wherein the coding region comprises atleast one modified uridine nucleoside, preferably selected fromN(1)-methylpseudouridine (m1ψ), pseudouridine (ψ), 5-methoxyuridine.Alternatively or in addition, the RNA as used herein comprises at leastone coding region as defined herein, wherein the coding region comprisesat least one modified cytosine nucleoside, preferably 5-methylcytosine.

Lipid Modification:

According to a further embodiment, a modified RNA as defined herein cancontain a lipid modification. Such a lipid-modified RNA as definedherein typically further comprises at least one linker covalently linkedwith that RNA, and at least one lipid covalently linked with therespective linker. Alternatively, the lipid-modified RNA comprises atleast one RNA as defined herein and at least one (bifunctional) lipidcovalently linked (without a linker) with that RNA. According to a thirdalternative, the lipid-modified RNA comprises an RNA molecule as definedherein, at least one linker covalently linked with that RNA, and atleast one lipid covalently linked with the respective linker, and alsoat least one (bifunctional) lipid covalently linked (without a linker)with that RNA. In this context, it is particularly preferred that thelipid modification is present at the terminal ends of a linear RNA.

Optimized Coding Sequences of the Original RNA:

The term “optimized coding sequence” relates to coding sequences thatdiffer in at least one codon (triplets of nucleotides coding for oneamino acid) compared to the corresponding wild type coding sequence.Preferably, a codon “optimized coding sequence” in the context of theinvention may show improved resistance to in vivo degradation and/orimproved stability in vivo, and/or improved translatability in vivo.Codon optimization in the broadest sense make use of the degeneracy ofthe genetic code wherein multiple codons may encode the same amino acidand may be used interchangeably to optimize/modify the coding sequencefor in vivo applications (e.g. vaccination).

In particularly preferred embodiments, the at least one RNA or at leastone RNA species is an mRNA comprising a coding region, wherein thecoding region of the original RNA sequence comprises an optimized codingsequence, wherein the optimized coding sequence is selected from Cmaximized coding sequence, G/C increased coding sequence, G/C optimizedcoding sequence, human codon usage optimized coding sequence, CAImaximized coding sequence, or any combination thereof.

According to certain embodiments, the RNA, particularly the codingsequence of the original RNA may be optimized, wherein the C content ofthe at least one coding sequence may be increased, preferably maximized,compared to the C content of the corresponding wild type coding sequence(herein referred to as “C maximized coding sequence”). The amino acidsequence encoded by the C maximized coding sequence of the nucleic acidsequence is preferably not modified as compared to the amino acidsequence encoded by the respective wild type nucleic acid codingsequence. The generation of a Cytosine optimized RNA as described abovemay suitably be carried out using a C maximization method according toWO2015/062738. In this context, the disclosure of WO2015/062738 isincluded herewith by reference.

According to some embodiments, the RNA, particularly the coding sequenceof the original RNA may be optimized, wherein the G/C content of thecoding sequence may be increased compared to the G/C content of thecorresponding wild type coding sequence (herein referred to as “G/Cincreased coding sequence”). The term “G/C increased coding sequence”relates to RNA, preferably the coding sequence of the original RNA ofthe invention that comprises an increased number of guanosine and/orcytosine nucleotides as compared to the corresponding wild type nucleicacid sequence. Such an increased number may be generated by substitutionof codons containing adenosine or thymidine nucleotides by codonscontaining guanosine or cytosine nucleotides. If the enriched G/Ccontent occurs in a coding sequence of DNA or RNA, it makes use of thedegeneracy of the genetic code. In particular, in case of RNA, sequenceshaving an increased G (guanosine)/C (cytosine) content are more stablethan sequences having an increased A (adenosine)/U (uracil) content. Theamino acid sequence encoded by the G/C content modified coding sequenceof the nucleic acid sequence is preferably not modified as compared tothe amino acid sequence encoded by the respective wild type nucleic acidcoding sequence. Preferably, the G/C content of the original RNA codingsequence of the present invention is increased by at least 10%,preferably by at least 20%, more preferably by at least 30%, mostpreferably by at least 40% compared to the G/C content of the codingsequence of the corresponding wild type nucleic coding sequence.

According to preferred embodiments, the RNA, particularly the codingsequence of the original RNA may be optimized, wherein the G/C contentof the coding sequence may be optimized compared to the G/C content ofthe corresponding wild type coding sequence (herein referred to as “G/Coptimized coding sequence”). “Optimized” in that context refers to acoding sequence wherein the G/C content is preferably increased to theessentially highest possible G/C content. The amino acid sequenceencoded by the G/C content optimized coding sequence of the nucleic acidsequence is preferably not modified as compared to the amino acidsequence encoded by the respective wild type nucleic acid codingsequence. The generation of a G/C optimized RNA sequences as describedabove may suitably be carried out using a G/C optimization methodexplained in WO2002/098443. In this context, the disclosure ofWO2002/098443 is included in its full scope in the present invention.

In particularly preferred embodiments, the at least one RNA or at leastone RNA species is an mRNA comprising a coding region, wherein the atleast one coding region of the original RNA sequence comprises a nucleicacid sequence, which is G/C optimized.

According to preferred embodiments, the RNA, particularly the codingsequence of the original RNA may be optimized, wherein the codons codingsequence may be optimized to the human codon usage (herein referred toas “human codon usage optimized coding sequence”). Codons encoding thesame amino acid occur at different frequencies in a subject, e.g. ahuman. Accordingly, the coding sequence of the RNA as defined herein ispreferably optimized such that the frequency of the codons encoding thesame amino acid corresponds to the naturally occurring frequency of thatcodon according to the human codon usage. For example, in the case ofthe amino acid Alanine (Ala), the wild type coding sequence ispreferably adapted in a way that the codon “GCC” is used with afrequency of 0.40, the codon “GCT” is used with a frequency of 0.28, thecodon “GCA” is used with a frequency of 0.22 and the codon “GCG” is usedwith a frequency of 0.10 etc. Accordingly, such a procedure (asexemplified for Ala) is applied for each amino acid encoded by thecoding sequence of the RNA, preferably the original RNA.

According to preferred embodiments, the RNA, particularly the codingsequence of the original RNA may be optimized, wherein the codonadaptation index (CAI) may be increased or preferably maximised (hereinreferred to as “CAI maximized coding sequence”). Accordingly, it ispreferred that all codons of the wild type sequence that are relativelyrare in the cell (e.g. a human) are exchanged for a respective codonthat is frequent in the cell, wherein the frequent codon encodes thesame amino acid as the relatively rare codon. Suitably, the mostfrequent codons are used for each encoded amino acid. Suitably, the RNA,preferably the original RNA of the present invention comprises at leastone coding sequence, wherein the codon adaptation index (CAI) of the atleast one coding sequence is at least 0.5, at least 0.8, at least 0.9 orat least 0.95. Most preferably, the codon adaptation index (CAI) of theat least one coding sequence is 1. For example, in the case of the aminoacid alanine (Ala) present in the amino acid sequence encoded by the atleast one coding sequence of the nucleic acid sequence according to theinvention, the wild type coding sequence is adapted in a way that themost frequent human codon “GCC” is always used for said amino acid.Accordingly, such a procedure (as exemplified for Ala) is applied foreach amino acid encoded by the coding sequence of the RNA, preferablythe original RNA to obtain CAI maximized coding sequences.

5′-cap Structure

According to another preferred embodiment of the invention, the RNA asused herein may be modified by the addition of a 5′-cap structure, whichpreferably stabilizes the RNA as described herein.

The term “5′-cap structure” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and is forexample intended to refer to a modified nucleotide (cap analogue),particularly a guanine nucleotide, added to the 5′ end of an RNAmolecule, e.g. an mRNA molecule. Preferably, the 5′-cap is added using a5′-5′-triphosphate linkage (also named m7GpppN). Further examples of5′-cap structures include glyceryl, inverted deoxy abasic residue(moiety), 4′,5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl)nucleotide, 4′-thio nucleotide, carbocyclic nucleotide,1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modifiedbase nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4′-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety,3′-3′-inverted a basic moiety, 3′-2′-inverted nucleotide moiety,3′-2′-inverted a basic moiety, 1,4-butanediol phosphate,3′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,3′phosphorothioate, phosphorodithioate, or bridging or non-bridgingmethylphosphonate moiety. Further modified 5′-cap structures which maybe used in the context of the present invention are cap1 (additionalmethylation of the ribose of the adjacent nucleotide of m7GpppN), cap2(additional methylation of the ribose of the 2nd nucleotide downstreamof the m7GpppN), cap3 (additional methylation of the ribose of the 3rdnucleotide downstream of the m7GpppN), cap4 (additional methylation ofthe ribose of the 4th nucleotide downstream of the m7GpppN), ARCA(anti-reverse cap analogue), modified ARCA (e.g. phosphothioate modifiedARCA), inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

A 5′ cap (cap0 or cap1) structure may also be formed in chemical RNAsynthesis or RNA in vitro transcription (co-transcriptional capping)using cap analogues.

The term “cap analogue” as used herein will be recognized and understoodby the person of ordinary skill in the art, and is for example intendedto refer to a non-polymerizable di-nucleotide that has cap functionalityin that it facilitates translation or localization, and/or preventsdegradation of a nucleic acid molecule, particularly of an RNA molecule,when incorporated at the 5′ end of the nucleic acid molecule.Non-polymerizable means that the cap analogue will be incorporated onlyat the 5′ terminus because it does not have a 5′ triphosphate andtherefore cannot be extended in the 3′ direction by a template-dependentpolymerase, particularly, by template-dependent RNA polymerase. Examplesof cap analogues include, but are not limited to, a chemical structureselected from the group consisting of m7GpppG, m7GpppA, m7GpppC;unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue(e.g., m2,7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG),dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or antireverse cap analogues (e.g., ARCA; m7,2′OmeGpppG, m7,2′dGpppG,m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives).Further cap analogues have been described previously (WO2008/016473,WO2008/157688, WO2009/149253, WO2011/015347, and WO2013/059475). Furthersuitable cap analogons in that context are described in WO2017/066793,WO2017/066781, WO2017/066791, WO2017/066789, WO2017/066782,WO2017/066797, wherein the disclosures referring to cap analogues areincorporated herewith by reference.

In a preferred embodiment, the 5′-cap structure is addedco-transcriptionally using cap-analogues, preferably as defined herein,in an RNA in vitro transcription reaction as described herein. Preferredcap-analogues in the context of the invention are m7G(5′)ppp(5′)G (m7G)or 3′-O-Me-m7G(5′)ppp(5′)G. Further preferred cap-analogues in thecontext of the invention are m7G(5′)ppp(5′)(2′OMeA)pG orm7G(5′)ppp(5′)(2′OMeG)pG to co-transcriptionally generate cap1structures.

In another embodiment, the 5′-cap structure is added via enzymaticcapping using capping enzymes (e.g. vaccinia virus capping enzymes,commercially available capping kits) to generate cap0 or cap1 or cap2structures. In other embodiments, the 5′-cap structure (cap0, cap1) isadded via enzymatic capping using immobilized capping enzymes, e.g. in acapping reactor (WO2016/193226).

m7GpppN (cap0), cap1 and cap2 are 5′-cap structure naturally occurringin RNA transcribed by polymerase II and is therefore not considered asmodification comprised in a modified RNA in this context. Accordingly, amodified RNA sequence of the present invention may comprise a cap0, cap1or cap2, but additionally the modified RNA sequence may comprise atleast one further modification as defined herein.

Poly(A) Sequence

According to another preferred embodiment of the invention, the RNA asused herein may contain a poly(A) sequence or a poly(A) tail.

The terms “poly(A) sequence”, “poly(A) tail” or “3′-poly(A) tail” arerecognized and understood by the person of ordinary skill in the art,and are for example intended to be a sequence of adenosine nucleotides,typically located at the 3′-end of an RNA, of up to about 400 adenosinenucleotides. In the context of the present invention, a poly(A) sequencemay be located within an mRNA or any other nucleic acid molecule, suchas, e.g., in a vector, for example, in a vector serving as template forthe generation of an RNA, preferably an mRNA, e.g., by transcription ofthe vector.

In a preferred embodiment, the poly(A) sequence, suitable located at the3′ terminus, is typically about 10 to 200 adenosine nucleotides,preferably about 10 to 100 adenosine nucleotides, more preferably about40 to 80 adenosine nucleotides or even more preferably about 50 to 70adenosine nucleotides. Preferably, the poly(A) sequence in the RNA asused herein is derived from a DNA template by RNA in vitrotranscription.

In other embodiments, the poly(A) sequence is obtained in vitro bycommon methods of chemical synthesis without being necessarilytranscribed from a DNA template.

In further embodiments, poly(A) sequences are generated by enzymaticpolyadenylation of the RNA (after RNA in vitro transcription) usingcommercially available polyadenylation kits and corresponding protocolsknown in the art, or alternatively, by using immobilized poly(A)polymerases, e.g. in a polyadenylation reactor (as described inWO2016174271).

Alternatively, the RNA as used herein may comprise a polyadenylationsignal. A polyadenylation signal typically comprises a hexamerconsisting of adenine and uracil/thymine nucleotides, preferably thehexamer sequence AAUAAA. Other sequences, preferably hexamer sequences,are also conceivable. Polyadenylation typically occurs during processingof a pre-mRNA (also called premature-mRNA). In this context, a consensuspolyadenylation signal is preferred comprising the NN(U/T)ANA consensussequence. In a particularly preferred embodiment, the polyadenylationsignal comprises one of the following sequences: AA(U/T)AAA orA(U/T)(U/T)AAA (wherein uridine is usually present in RNA and thymidineis usually present in DNA).

In some embodiments, the RNA as used herein may contain a poly(A)sequence derived from a vector and at least one additional poly(A)sequence generated by enzymatic polyadenylation, e.g. as described inWO2016/091391.

Poly(C) Sequence

According to a further preferred embodiment, the RNA as used herein maycontain a poly(C) sequence. The term “poly(C) sequence” has to beunderstood as a long sequence of cytosine nucleotides, typically about10 to about 200 cytosine nucleotides, preferably about 10 to about 100cytosine nucleotides, more preferably about 10 to about 70 cytosinenucleotides or even more, preferably about 20 to about 50, or even about20 to about 30 cytosine nucleotides. A poly(C) sequence may preferablybe located 3′ of the coding sequence comprised by a nucleic acid.Preferably, the poly(C) sequence in the RNA as used herein is derivedfrom a DNA template by RNA in vitro transcription.

UTRs

In a preferred embodiment, the at least one RNA or the at least one RNAspecies comprises a 3′-UTR and/or a 5′-UTR. Preferably, a 3′-UTR or a5′-UTR as used herein comprises one or more 3′-UTR elements or 5′-UTRelements, respectively. Accordingly, the RNA as used herein may compriseat least one 5′-UTR element and/or at least one 3′-UTR element. In thiscontext, an UTR element comprises or consists of a nucleic acidsequence, which is derived from a 5′-UTR or 3′-UTR of any naturallyoccurring gene or which is derived from a fragment, a homolog or avariant of the 5′-UTR or 3′-UTR of a gene. Preferably, the 5′-UTR or3′-UTR element used in the context of the present invention isheterologous to the at least one coding sequence of the RNA as usedherein. 5′-UTR or 3′-UTR elements are preferably derived from naturallyoccurring genes. In other embodiments, synthetically engineered 5′-UTRor 3′-UTR elements may be used in the context of the present invention.

The term “3′-untranslated region (3′-UTR)” will be recognized andunderstood by the person of ordinary skill in the art, and are forexample intended to refer to a part of a nucleic acid molecule, which islocated 3′ (i.e. “downstream”) of a coding sequence and which istypically not translated into protein. Usually, a 3′-UTR is the part ofan mRNA which is located between the coding sequence (cds) and thepoly(A) sequence of the mRNA. In the context of the invention, the term3′-UTR may also comprise elements, which are not encoded in the DNAtemplate, from which RNA is transcribed, but which are added aftertranscription during maturation, e.g. a poly(A) sequence.

The term “5′-untranslated region (5′-UTR)” will be recognized andunderstood by the person of ordinary skill in the art, and are forexample intended to refer to a part of a nucleic acid molecule, which islocated 5′ (i.e. “upstream”) of a coding sequence and which is nottranslated into protein. A 5′-UTR is typically understood to be aparticular section of messenger RNA (mRNA), which is located 5′ of thecoding sequence of the mRNA. Typically, the 5′-UTR starts with thetranscriptional start site and ends one nucleotide before the startcodon of the coding sequence. Preferably, the 5′-UTRs have a length ofmore than 20, 30, 40 or 50 nucleotides. The 5′-UTR may comprise elementsfor controlling gene expression, also called regulatory elements. Suchregulatory elements may be, for example, ribosomal binding sites. The5′-UTR may be post-transcriptionally modified, for example by additionof a 5′-cap.

3′-UTR Elements

In a preferred embodiment, the RNA as used herein may comprise at leastone 3′-UTR element, which is typically located within the 3′-UTR of theRNA as described herein.

Preferably, the 3′-UTRs in the context of the invention are heterologousto the coding sequence. More preferably, the RNA as used herein,preferably the 3′UTR of the RNA as used herein, comprises at least oneheterologous 3′-UTR element.

Preferably, the at least one 3′-UTR element comprises or consists of anucleic acid sequence derived from the 3′-UTR of a chordate gene,preferably a vertebrate gene, more preferably a mammalian gene, mostpreferably a human gene, or from a variant of the 3′-UTR of a chordategene, preferably a vertebrate gene, more preferably a mammalian gene,most preferably a human gene.

Preferably, the RNA as used herein comprises a 3′-UTR element,preferably as described herein, which may be derivable from a gene thatrelates to RNA with an enhanced half-life (that provides a stable RNA).Preferably, the 3′ UTR element is a nucleic acid sequence derived from a3′ UTR of a gene, which preferably encodes a stable RNA, or from ahomolog, a fragment or a variant of said gene

In a particularly preferred embodiment, the 3′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 3′-UTR of agene selected from the group consisting of an albumin gene, analpha-globin gene, a beta-globin gene, a tyrosine hydroxylase gene, alipoxygenase gene, and a collagen alpha gene, such as a collagen alpha1(I) gene, or from a variant of a 3′-UTR of a gene selected from thegroup consisting of an albumin gene, an alpha-globin gene, a beta-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagenalpha gene, such as a collagen alpha 1(I) gene according to SEQ ID NOs:1369-1393 of the patent application WO2013/143700, or from a homolog, afragment or a variant thereof. Particularly preferred are 3′-UTRsequences according to SEQ ID NO: 1369 (Human albumin 3′-UTR) and SEQ IDNO: 1376 (albumin7 3′-UTR) of the patent application WO2013/143700. Inthis context, the disclosure of WO2013/143700 is incorporated herein byreference. Alternatively, the RNA as used herein preferably comprises a3′-UTR element, which comprises or consists of a nucleic acid sequenceselected from SEQ ID NOs: 30572, 30574, 30576, 30578, 30580, 30582 or30584 or a fragment or variant thereof.

In other embodiments, the RNA as used herein comprises a 3′-UTR elementaccording to any one of SEQ ID NOs: 10 to 205 of WO2015/101414 and SEQID NO: 1 and 2 of WO2015/101415. In this context, the disclosures ofWO2015/101414 and WO2015/101415 are incorporated herewith by reference.

In some embodiments, the RNA used herein comprises a 3′-UTR element,which may be any 3′-UTR element described in WO2016/107877. In thiscontext, the disclosure of WO2016/107877 relating to 3′-UTRelements/sequences is herewith incorporated by reference. Particularlypreferred 3′-UTR elements are SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to318 of the patent application WO2016/107877, or fragments or variants ofthese sequences. In this context, it is particularly preferred that the3′-UTR element of the RNA sequence according to the present inventioncomprises or consists of a corresponding RNA sequence of the nucleicacid sequence according SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to 318 ofthe patent application WO2016/107877.

In certain embodiments, the RNA as defined herein comprises a 3′-UTRelement, which may be any 3′-UTR element as described in WO2017/036580.In this context, the disclosure of WO2017/036580 relating to 3′-UTRelements/sequences is herewith incorporated by reference. Particularlypreferred 3′-UTR elements are nucleic acid sequences according to SEQ IDNOs: 152 to 204 of the patent application WO2017/036580, or fragments orvariants of these sequences. In this context, it is particularlypreferred that the 3′-UTR element of the RNA sequence according to thepresent invention comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according SEQ ID NOs: 152 to 204 of thepatent application WO2017/036580.

Further, particularly suitable 3′-UTRs are disclosed in WO2018172556.The disclosure of WO2018172556, in particular relating to GNAS, CASP1,PSMB3, ALB, COX6B1, NDUFA1 and RPS9, is included herewith by reference.Preferred 3′-UTR elements are derived from GNAS, CASP1, PSMB3, ALB,COX6B1, NDUFA1 or RPS9. Particularly preferred 3′-UTR elements arenucleic acid sequences according to SEQ ID NOs: 23 to 36 as disclosed inWO2018172556, or fragments or variants of these sequences.

5′-UTR Elements

In a preferred embodiment, the RNA as used herein may comprise at leastone 5′-UTR element, which is typically located within the 5′-UTR of theRNA as described herein.

Preferably, the 5′-UTRs in the context of the invention are heterologousto the coding sequence. More preferably, the RNA as used herein,preferably the 5′UTR of the RNA as used herein, comprises at least oneheterologous 5′-UTR element.

In preferred embodiments, the RNA as used herein comprises at least one5′-UTR element. Preferably, the at least one 5′-UTR element comprises orconsists of a nucleic acid sequence derived from the 5′-UTR of achordate gene, preferably a vertebrate gene, more preferably a mammaliangene, most preferably a human gene, or from a variant of the 5′-UTR of achordate gene, preferably a vertebrate gene, more preferably a mammaliangene, most preferably a human gene.

In certain embodiments, the RNA as used herein comprises at least one5′-UTR element, which may be any 5′-UTR element as described in thepatent application WO2013/143700. Suitably, the at least one 5′-UTRelement is derived from a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 andSEQ ID NO: 1422 of the patent application WO2013/143700. In thiscontext, the disclosure of WO2013/143700 is incorporated herewith byreference.

In preferred embodiments, the RNA as used herein comprises at least oneheterologous 5′-UTR element, which comprises or consists of a nucleicacid sequence, which is derived from the 5′-UTR of a TOP gene,preferably from a corresponding RNA sequence, or a homolog, a fragment,or a variant thereof, preferably lacking the 5′TOP motif. Morepreferably, the at least one heterologous 5′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 5′-UTR of aTOP gene encoding a ribosomal protein, preferably from a correspondingRNA sequence, or a homolog, a fragment or a variant of said nucleic acidsequence, preferably lacking the 5′TOP motif

In preferred embodiments, the at least one 5′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 5′-UTR of aTOP gene encoding a ribosomal protein or from a variant of a 5′-UTR of aTOP gene encoding a ribosomal protein. For example, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 5′-UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67,170, 193, 244, 259, 554, 650, 675, 700, 721, 913, 1016, 1063, 1120,1138, and 1284-1360 of the patent application WO2013/143700, acorresponding RNA sequence, a homolog thereof, or a variant thereof asdescribed herein, preferably lacking the 5′-TOP motif.

In some embodiments, the at least one 5′-UTR element, preferably aheterologous 5′-UTR element, comprises or consists of a nucleic acidsequence, which is derived from a 5′-UTR of a TOP gene encoding aribosomal Large protein (RPL), preferably from RPL32 or RPL35A, or froma gene selected from the group consisting of HSD17B4, ATP5A1, AIG1,COXC6 or ABCB7(MDR), or from a homolog, a fragment or variant of any oneof these genes, preferably lacking the 5′TOP motif.

Preferably, the 5′-UTR element comprises or consists of a nucleic acidsequence, which is derived from a 5′-UTR of a TOP gene encoding aribosomal Large protein (RPL) or from a homolog or variant of a 5′-UTRof a TOP gene encoding a ribosomal Large protein (RPL). For example, the5′-UTR element comprises or consists of a nucleic acid sequence, whichis derived from a 5′-UTR of a nucleic acid sequence according to any ofSEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421and 1422 of the patent application WO2013/143700, a corresponding RNAsequence, a homolog thereof, or a variant thereof as described herein,preferably lacking the 5′-TOP motif.

In a preferred embodiment, the 5′-UTR element comprises or consists of anucleic acid sequence, according SEQ ID NO: 1368 of the patentapplication WO2013/143700, or preferably to a corresponding RNAsequence, or fragments or variants thereof. In another preferredembodiment, the 5′-UTR element comprises or consists of a nucleic acidsequence according to SEQ ID NOs: 1412-1420 of the patent applicationWO2013/143700, or a corresponding RNA sequence, or fragments or variantsthereof.

Alternatively, the 5′-UTR element preferably comprises or consists of anucleic acid sequence according to SEQ ID NO: 30570 as disclosed herein,or a fragment or variant thereof.

In some embodiments, the RNA as used herein comprises a 5′-UTR element,which may be any 5′-UTR element described in WO2016/107877. In thiscontext, the disclosure of WO2016/107877 relating to 3′-UTRelements/sequences is herewith incorporated by reference. Particularlypreferred 5′-UTR elements are nucleic acid sequences according to SEQ IDNOs: 25 to 30 and SEQ ID NOs: 319 to 382 of the patent applicationWO2016/107877, or fragments or variants of these sequences. In thiscontext, it is particularly preferred that the 5′-UTR element of the RNAsequence according to the present invention comprises or consists of acorresponding RNA sequence of the nucleic acid sequence according SEQ IDNOs: 25 to 30 and SEQ ID NOs: 319 to 382 of the patent applicationWO2016/107877.

In some embodiments, the RNA as used herein comprises a 5′-UTR element,which may be any 5′-UTR element as described in WO2017/036580. In thiscontext, the disclosure of WO2017/036580 relating to 3′-UTRelements/sequences is herewith incorporated by reference. Particularlypreferred 5′-UTR elements are nucleic acid sequences according to SEQ IDNOs: 1 to 151 of the patent application WO2017/036580, or fragments orvariants of these sequences. In this context, it is particularlypreferred that the 5′-UTR element of the RNA sequence according to thepresent invention comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID NOs: 1 to 151 of thepatent application WO2017/036580.

Further, particularly suitable 5′-UTRs are disclosed in WO2018172556.The disclosure of WO2018172556, in particular relating to SLC7A3,ATP5A1, RPL32, HSD17B4, NOSIP, ASAH1, RPL31, TUBB4B, UBQLN2, MP68 andNDUFA4, is included herewith by reference. Preferred 3′-UTR elements arederived from SLC7A3, ATP5A1, RPL32, HSD17B4, NOSIP, ASAH1, RPL31,TUBB4B, UBQLN2, MP68 or NDUFA4. Particularly preferred 3′-UTR elementsare nucleic acid sequences according to SEQ ID NOs: 1 to 22 as disclosedin WO2018172556, or fragments or variants of these sequences.

Preferably, the at least one 5′-UTR element as defined herein and the atleast one 3′-UTR element as defined herein act synergistically toincrease protein production from the at least one RNA sequence asdescribed above.

Histone Stem-Loop

In a particularly preferred embodiment, the RNA as used herein comprisesa histone stem-loop sequence/structure. The term “histone stem-loop” asused herein will be recognized and understood by the person of ordinaryskill in the art, and are for example intended to refer to nucleic acidsequences that are predominantly found in histone histone mRNAs.Exemplary histone stem-loop sequences are described in Lopez et al.(Davila Lopez, M., & Samuelsson, T. (2008), RNA, 14(1)). The stem-loopsin histone pre-mRNAs are typically followed by a purine-rich sequenceknown as the histone downstream element (HDE). These pre-mRNAs areprocessed in the nucleus by a single endonucleolytic cleavageapproximately 5 nucleotides downstream of the stem-loop, catalyzed bythe U7 snRNP through base pairing of the U7 snRNA with the HDE.

Such histone stem-loop sequences are preferably selected from histonestem-loop sequences as disclosed in WO2012/019780, the disclosurerelating to histone stem-loop sequences/structures incorporated herewithby reference.

A histone stem-loop sequence suitable to be used within the presentinvention is preferably derived from formulae (I) or (II) of the patentapplication WO2012/019780. According to a further preferred embodimentthe RNA as defined herein may comprise at least one histone stem-loopsequence derived from at least one of the specific formulae (Ia) or(IIa) of the patent application WO2012/019780.

A particular preferred histone stem-loop sequence is the nucleic acidsequence according to SEQ ID NO: 30586 disclosed herein, or the sequenceCAAAGGCTCTTTTCAGAGCCACCA (according to SEQ ID NO: 43 of the patentapplication WO2015024667) or more preferably the corresponding RNAsequence CAAAGGCUCUUUUCAGAGCCACCA (according to SEQ ID NO: 44 of thepatent application WO2015024667).

Any of the above modifications may be applied to the RNA (or RNAspecies) in the context of the present invention and may be, if suitableor necessary, be combined with each other in any combination, provided,these combinations of modifications do not interfere with each other inthe respective nucleic acid sequence. A person skilled in the art willbe able to take his choice accordingly.

Accordingly, the RNA as used herein may comprise a 5′-UTR and/or a3′-UTR preferably comprising at least one histone stem-loop. The 3′-UTRof the RNA as used herein preferably comprises also a poly(A) and/or apoly(C) sequence, preferably as defined herein. The single elements ofthe 3′ UTR may occur therein in any order from 5′ to 3′ along thesequence of the RNA sequence of the present invention. In addition,further elements as described herein, may also be contained, such as astabilizing sequence as defined herein (e.g. derived from the UTR of aglobin gene), IRES sequences, miRNA binding sites etc. Each of theelements may also be repeated in the RNA as used herein at least once(particularly in di- or multicistronic constructs), preferably twice ormore.

In a preferred embodiment, the RNA (RNA species) as used herein ismonocistronic, bicistronic or multicistronic. Preferably, the RNAcomprises at least two coding regions and at least one IRES sequence.

Accordingly, in a preferred embodiment, the RNA as used hereincomprises, preferably in 5′- to 3′-direction:

a) a 5′-cap structure (cap0, cap1, cap2), preferably m7GpppN (cap0);b) optionally, a 5′-UTR element as defined herein;c) at least one coding sequence;d) a 3′-UTR element as defined herein;e) optionally, a poly(A)sequence, preferably comprising 10 to 200, 10 to100, 40 to 80 or 50 to 70 adenine nucleotides, more preferablycomprising 64 adenine nucleotides;f) optionally, a poly(C)sequence, preferably consisting of 10 to 200, 10to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, morepreferably comprising 30 cytosine nucleotides;g) optionally, a histone stem-loop;h) optionally, an additional poly(A) sequence;or a fragment or variant of any of these nucleic acid sequences.

In certain embodiments, the RNA (or RNA species) as used hereincomprises, preferably in 5′ to 3′ direction, the following elements:

a) a 5′-cap structure, preferably m7GpppN,b) at least one coding region;c) a 3′-UTR element comprising a nucleic acid sequence, which is derivedfrom an alpha-globin gene, preferably comprising the nucleic acidsequence according to SEQ ID NO: 30578, or a homolog, a fragment or avariant of any one of these nucleic acid sequences,d) a poly(A)sequence, preferably comprising 10 to 200, 10 to 100, 40 to80 or 50 to 70 adenine nucleotides, more preferably comprising 64adenine nucleotides,e) a poly(C)sequence, preferably consisting of 10 to 200, 10 to 100, 20to 70, 20 to 60 or 10 to 40 cytosine nucleotides, more preferablycomprising 30 cytosine nucleotides, andf) a histone stem-loop, preferably comprising the nucleic acid sequenceaccording to SEQ ID NO. 30586, or a fragment or variant of any one ofthese nucleic acid sequences.

According to some preferred embodiments, the RNA (or RNA species) asused herein comprises, preferably in 5′ to 3′ direction, the followingelements:

a) a 5′-cap structure, preferably m7GpppN,b) a 5′-UTR element, which comprises a nucleic acid sequence, which isderived from the 5′-UTR of a TOP gene, preferably comprising a nucleicacid sequence according to SEQ ID NO: 30570, or a homolog, a fragment ora variant of any one of these nucleic acid sequences,c) at least one coding region,d) a 3′-UTR element comprising a nucleic acid sequence, which is derivedfrom an albumin gene, preferably comprising the corresponding RNAsequence of the nucleic acid sequence according to SEQ ID NO: 30582 or30584, or a homolog, a fragment or a variant of any one of these nucleicacid sequences,e) a poly(A)sequence, preferably comprising 10 to 200, 10 to 100, 40 to80 or 50 to 70 adenine nucleotides, more preferably comprising 64adenine nucleotides,f) a poly(C)sequence, preferably consisting of 10 to 200, 10 to 100, 20to 70, 20 to 60 or 10 to 40 cytosine nucleotides, more preferablycomprising 30 cytosine nucleotides, andg) a histone stem-loop, preferably comprising the nucleic acid sequenceaccording to SEQ ID NO. 30586, or a fragment or variant of any one ofthese nucleic acid sequences.

The at least one coding region (or coding sequence) of the RNA (or RNAspecies) that is adapted according to the methods disclosed herein,preferably encodes a peptide or a protein.

In preferred embodiments, the at least one coding region encodes anantigen derived from a pathogen causing an infectious disease, or afragment or variant of such an antigen. More preferably, the at leastone coding region encodes a viral antigen, a bacterial antigen, a fungalantigen or a protozoan antigen, or a fragment or variant of any one ofthese antigens.

In this context, further preferred are antigens derived from pathogensselected from the group consisting of Acinetobacter baumannii, Anaplasmagenus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostomaduodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides,Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis,Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis,Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi,Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi,Bunyaviridae family, Burkholderia cepacia and other Burkholderiaspecies, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridaefamily, Campylobacter genus, Candida albicans, Candida spp, Chlamydiatrachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJDprion, Clonorchis sinensis, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, Clostridium perfringens, Clostridiumspp, Clostridium tetani, Coccidioides spp, coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus,Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4),Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichiachaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica,Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie Avirus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus(EBV), Escherichia coli O157:H7, O111 and O104:H4, Fasciola hepatica andFasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,Francisella tularensis, Fusobacterium genus, Geotrichum candidum,Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis BVirus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis EVirus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasmacapsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Humanbocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7(HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus,Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassavirus, Legionella pneumophila, Leishmania genus, Leptospira genus,Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV),Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Naegleria fowleri, Necator americanus, Neisseriagonorrhoeae, Neisseria meningitidis, Norovirus, Nocardia asteroides,Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi,Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis,Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurellagenus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabiesvirus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses,Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsiarickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus,Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARScoronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus,Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcusgenus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium,Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati,Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonasvaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei,Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV),Varicella zoster virus (VZV), Variola major or Variola minor, vCJDprion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nilevirus, Western equine encephalitis virus, Wuchereria bancrofti, Yersiniaenterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis,preferably the pathogen is selected from the group consisting ofinfluenza virus, respiratory syncytial virus (RSV), Herpes simplex virus(HSV), human Papilloma virus (HPV), Human immunodeficiency virus (HIV),Plasmodium, Staphylococcus aureus, Dengue virus, Chlamydia trachomatis,Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacteriumtuberculosis, Rabies virus, Rotavirus and Yellow Fever Virus, Zikavirus.

According to a particularly preferred embodiment, the at least onecoding region encodes an antigen, or a fragment or variant thereof,derived from an influenza virus, more preferably from an influenza A, aninfluenza B, or an influenza C virus (strain) or a variant of any ofthese.

In this context it, is particularly preferred that the at least onecoding region of the RNA used herein encodes at least one antigen, or afragment or variant thereof, selected from the group consisting ofhemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrixprotein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1),non-structural protein 2 (NS2), nuclear export protein (NEP), polymeraseacidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerasebasic protein 2 (PB2) of an influenza virus or a variant thereof. Insome embodiments, the at least one coding region of the RNA used hereinencodes at least one antigenic peptide or protein, or a fragment orvariant thereof, which is derived from any one of the influenza virusproteins mentioned herein.

In a preferred embodiment, the at least one coding region of the RNAused herein encodes at least one antigen selected from hemagglutinin(HA) and/or neuraminidase (NA) of an influenza virus, or a fragment orvariant thereof. More preferably, the at least one coding region of theRNA used herein encodes at least one antigenic peptide or proteinderived from hemagglutinin (HA) and/or neuraminidase (NA) of aninfluenza virus, or a fragment or variant thereof. In this context, thehemagglutinin (HA) and the neuraminidase (NA) may be chosen from thesame influenza virus or from different influenza viruses (or differentinfluenza virus strains, respectively).

Influenza antigens and antigenic peptides or proteins derived frominfluenza virus are described in international patent applicationsPCT/EP2016/075862 and PCT/EP2017/060663, which are hereby incorporatedby reference in their entirety.

In preferred embodiments, the RNA (species) as used herein, preferablythe original RNA (species) that is to be adapted according to themethods disclosed herein or the adapted RNA (species), comprises atleast one coding region, which encodes a peptide or protein comprisingor consisting of an amino acid sequence according to any one of SEQ IDNOs: SEQ ID NOs: 1-30504, 213713, 213738, 213739, 213787, 213792,213797, 213802, 213996-214023, 214100-214127, 214212-214239,214316-214343, 214420-214447, 214524-214551, 214628-214655,214732-214759, 214836-214863, 214940-214967, 215044, 215049-215076,215161, 215166-215193, 215278, 215283-215310, 215395, 215400-215427,215512, 215517-215544 as described in PCT/EP2017/060663, or a fragmentor variant of any one of these amino acid sequences.

According to some embodiments, the RNA (species) used herein, preferablythe original RNA (species) that is to be adapted, comprises at least onecoding region comprising or consisting of a nucleic acid sequenceaccording to any one of SEQ ID NOs: 30505-213528, 213529-213557,213740-213746, 213788, 213789, 213793, 213794, 213798, 213799, 213803,213804, 214024-214051, 214128-214155, 214240-214267, 214344-214371,214448-214475, 214552-214579, 214656-214683, 214760-214787,214864-214891, 214968-214995, 215045, 215046, 215077-215104, 215162,215163, 215194-215221, 215279, 215280, 215311-215338, 215396, 215397,215428-215455, 215513, 215514, 215545-215572, 215629, 215632,215638-215835, 215892, 215836-215889 as described in PCT/EP2017/060663,or a fragment or variant of any one of these nucleic acid sequences.

According to a further preferred embodiment, the RNA (species) usedherein comprises at least one coding region encoding an antigen, or afragment or variant thereof, derived from a Norovirus, preferablyselected from a GI.1 to GI.17 Norovirus, GII.1 to GII.24 Norovirus,GIII.1 to GIII.4 Norovirus, GIV.1 to GIV.4 Norovirus and GV.1 to GV.4Norovirus more preferably, from a Norovirus selected from the groupconsisting of GI.1 Norovirus and GII.4 Norovirus or a variant of any ofthese.

In this context it, is particularly preferred that the at least onecoding region of the RNA used herein encodes at least one antigen, or afragment or variant thereof, selected from the group consisting ofNorovirus non-structural proteins NS1/NS2, NS3, NS4, NS5, NS6, NS7,Norovirus capsid protein VP1 and Norovirus capsid protein VP2 or avariant thereof. In some embodiments, the at least one coding region ofthe RNA used herein encodes at least one antigenic peptide or protein,or a fragment or variant thereof, which is derived from any one of theNorovirus proteins mentioned herein.

In a preferred embodiment, the at least one coding region of the RNAused herein encodes at least one antigen selected from Norovirus capsidprotein VP1 or Norovirus capsid protein VP2 or a fragment or variantthereof. In this context, the Norovirus capsid protein VP1 and/orNorovirus capsid protein VP2 may be chosen from the same Norovirus orfrom different Noroviruses (or different Norovirus strains,respectively). Norovirus antigens and antigenic peptides or proteinsderived from Norovirus are also described in international patentapplication PCT/EP2017/060673, which is hereby incorporated by referencein its entirety.

In preferred embodiments, the RNA (species) as used herein, preferablythe original RNA (species) that is to be adapted according to themethods disclosed herein or the adapted RNA (species), comprises atleast one coding region, which encodes a peptide or protein (derivedfrom a Norovirus) comprising or consisting of an amino acid sequenceaccording to any one of SEQ ID NOs: 1-4410 as described inPCT/EP2017/060673, or a fragment or variant of any one of these aminoacid sequences.

According to some embodiments, the RNA (species) used herein, preferablythe original RNA (species) that is to be adapted, comprises at least onecoding region comprising or consisting of a nucleic acid sequenceaccording to any one of SEQ ID NOs: 4411-39690, 39713-39746 as describedin PCT/EP2017/060673, or a fragment or variant of any one of thesenucleic acid sequences.

According to some embodiments, the RNA (species) used herein, preferablythe original RNA (species) that is to be adapted or the adapted RNA(species), comprises at least one coding region encoding a tumorantigen. Suitably, the tumor antigen is selected from the listconsisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m,alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1,BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsinB, cathepsin L, CD1, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55,CD56, CD80, CDCl27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66,COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m,EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2,FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE7b, GAGE-8, GDEP, GnT-V, gpl OO, GPC3, GPNMB/m, HAGE, HAST-2,hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R1 71, HLA-A1 1/m, HLA-A2/m,HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7,HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immaturelaminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205,KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-Al2, MAGE-B1,MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16,MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-El,MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A,MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, MEI/m, mesothelin,MG50/PXDN, MMP1 1, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m,MUM-2/m, MUM-3/m, myosin class l/m, NA88-A,N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP,NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT,OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, pi 5, p190 minor bcr-abl,p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase,Pin-1, Pml/PARalpha, POTE, PRAME, PRDXS/m, prostein, proteinase-3, PSA,PSCA, PSGR, PSM, PSMA, PTPRK m, RAGE-1, RBAF600/m, RHAMM/CD1 68, RU1,RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1,SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B,SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta,TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2,TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, and WT1.

In another particularly preferred embodiment, the RNA (species) usedherein, preferably the original RNA (species) that is to be adapted orthe adapted RNA (species), comprises at least one coding regioncomprising or consisting of a nucleic acid sequence encoding at leastone therapeutic peptide or protein which can be used inter alia in thetreatment of e.g. metabolic or endocrine disorders. These and otherproteins are understood to be therapeutic, as they are meant to treatthe subject by replacing its defective endogenous production of afunctional protein in sufficient amounts. Accordingly, such therapeuticproteins are typically mammalian, in particular human proteins.

Preferably, the therapeutic peptide or protein is selected from orderived from any therapeutic peptide or protein which is used or can beused for medical treatment (e.g. protein replacement), antibodies, Tcell receptors, gene-editing proteins (e.g. Cas9).

In another particularly preferred embodiment, the RNA (species) as usedherein, preferably the original RNA (species) that is to be adapted orthe adapted RNA (species), encodes at least one therapeutic antibodyselected from the group consisting of antibodies which are used interalia for the treatment of cancer or tumor diseases, immune disorders,infectious diseases, Alzheimer's disease, asthma, and antibodies whichare used for the treatment of diverse disorders, e.g. osteoporosis,tooth decay, idiopathic pulmonary fibrosis, focal segmentalglomerulosclerosis, pain, muscular dystrophy, and Neovascularage-related macular degeneration.

In another particularly preferred embodiment the RNA (species) usedherein, preferably the original RNA (species) that is to be adapted orthe adapted RNA (species), comprises at least one coding regioncomprising or consisting of a nucleic acid sequence encoding at leastone allergen associated with allergy or an allergic disease (allergensor allergenic antigens) and/or at least one autoimmune self-antigens(autoantigens).

Adapted RNA and Vectors:

In a further aspect, the present invention relates to the RNA that isobtainable by the methods according to the invention. More specifically,the invention provides an adapted RNA (or an RNA (species) with anadapted sequence, respectively), which is obtainable by the method formodifying the retention time of an RNA on a chromatographic column, bythe method for purifying at least one RNA species from a mixture of atleast two RNA species, by the method for co-purifying at least two RNAspecies from a mixture comprising at least two RNA species, by themethod for harmonizing the numbers of A and/or U nucleotides in thesequences of at least two RNA species, or by the method for providing anadapted RNA as described herein.

The (adapted) RNA according to the invention is preferably characterizedin that its sequence was adapted by altering the number of A and/or Unucleotides with respect to the number of A and/or U nucleotides in theoriginal RNA sequence. The original RNA sequence is preferably an RNAsequence as described herein, preferably the corresponding wild-type RNAsequence, more preferably a corresponding RNA sequence that was modifiedas described herein, e.g. G/C content modified or codon optimized.

In preferred embodiments, the adapted RNA according to the invention ischaracterized by any one of the features described herein, in particularwith respect to the inventive methods.

In certain embodiments, the adapted RNA according to the invention ischaracterized by a ratio of the number of A nucleotides to the number ofU nucleotides in the RNA sequence, which is in the range from 0.2 to 5,preferably from 0.5 to 3, more preferably from 1 to 3, even morepreferably from 1 to 2.5, even more preferably from 1.2 to 2, even morepreferably from 1.4 to 2, even more preferably from 1.5 to 2, mostpreferably from 1.6 to 2.

According to a particularly preferred embodiment, the RNA according tothe invention comprises at least one coding region comprising orconsisting of a nucleic acid sequence according to any one of SEQ ID NO:26 to 14079, 14080 to 16264, 16265 to 28640, 28641 to 30568, or afragment or variant of any one of these nucleic acid sequences.

In a further aspect, the present invention concerns a vector comprisingthe adapted RNA sequence according to the invention. In preferredembodiments, the vector according to the invention is a DNA vectorcomprising a nucleic acid sequence corresponding to the adapted RNAsequence according to the invention, or a fragment or variant thereof.Alternatively, the vector according to the invention is a DNA vectorcomprising a nucleic acid sequence encoding the adapted RNA sequenceaccording to the invention, or a fragment or variant thereof.Preferably, the vector according to the invention is a plasmid vector ora viral vector.

RNA Production

The RNA according to the present invention may be prepared using anymethod known in the art, including synthetic methods such as e.g. solidphase RNA synthesis, as well as in vitro methods, such as RNA in vitrotranscription reactions. Particularly preferred is RNA in vitrotranscription.

The terms “RNA in vitro transcription” or “in vitro transcription”relate to a process, wherein RNA is synthesized in a cell-free system(in vitro). DNA, particularly plasmid DNA (or PCR product), is typicallyused as template for the generation of RNA transcripts. RNA may beobtained by DNA-dependent in vitro transcription of an appropriate DNAtemplate, which according to the present invention is preferably alinearized plasmid DNA template. The promoter for controlling in vitrotranscription can be any promoter for any DNA-dependent RNA polymerase.Particular examples of DNA-dependent RNA polymerases are the T7, T3, andSP6 RNA polymerases. A DNA template for in vitro RNA transcription maybe obtained by cloning of a nucleic acid, in particular cDNAcorresponding to the respective RNA to be in vitro transcribed, andintroducing it into an appropriate vector for in vitro transcription,for example into plasmid DNA. In a preferred embodiment of the presentinvention the DNA template is linearized with a suitable restrictionenzyme, before it is transcribed in vitro. The cDNA may be obtained byreverse transcription of mRNA or chemical synthesis. Moreover, the DNAtemplate for in vitro RNA synthesis may also be obtained by genesynthesis.

Reagents used in RNA in vitro transcription typically include: a DNAtemplate (linearized plasmid DNA or PCR product) with a promotersequence that has a high binding affinity for its respective RNApolymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6,or Syn5); ribonucleoside triphosphates (NTPs) for the four bases(adenine, cytosine, guanine and uracil); optionally, a cap analogue asdefined herein (e.g. m7G(5′)ppp(5′)G (m7G)); optionally, furthermodified nucleotides as defined herein; a DNA-dependent RNA polymerasecapable of binding to the promoter sequence within the DNA template(e.g. T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease(RNase) inhibitor to inactivate any contaminating RNase; optionally, apyrophosphatase to degrade pyrophosphate, which may inhibittranscription; MgCl2, which supplies Mg2+ ions as a co-factor for thepolymerase; a buffer (Tris or HEPES) to maintain a suitable pH value,which can also contain antioxidants (e.g. DTT), and/or polyamines suchas spermidine at optimal concentrations, or a buffer system as disclosedin WO2017/109161.

In embodiments, the nucleotide mixture used in RNA in vitrotranscription may additionally contain modified nucleotides as definedherein. In embodiments, the nucleotide mixture (i.e. the fraction ofeach nucleotide in the mixture) may be optimized for the given RNAsequence, preferably as described WO2015/188933.

In embodiment where more than one different RNA as defined herein has tobe produced, e.g. where 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent RNA molecules have to be produced, procedures as described inWO2017/109134 may be suitably used.

In a preferred embodiment, RNA production is performed under currentgood manufacturing practice (GMP), implementing various quality controlsteps on DNA and RNA level, according to WO2016/180430. The obtained RNAproducts are preferably purified using PureMessenger® (CureVac,TUbingen, Germany; RP-HPLC according to WO2008/077592) and/or tangentialflow filtration (as described in WO2016/193206). In a preferredembodiment, the RNA, particularly the purified RNA is lyophilizedaccording to WO2016/165831 or WO2011/069586 to yield a temperaturestable RNA as defined herein. The RNA of the invention, particularly thepurified RNA may also be lyophilized using spray-drying or spray-freezedrying according to WO2016/184575 or WO2016184576 to yield a temperaturestable RNA as defined herein.

Method for Providing an Adapted RNA Sequence

According to a further aspect, the invention also provides a method forproviding an adapted RNA sequence, wherein the method preferablycomprises the steps of

a) optionally, determining the total number of nucleotides in anoriginal RNA sequence;b) determining the number of A and/or U nucleotides in the original RNAsequence;c) determining the codons in the original RNA sequence that can bereplaced with at least one alternative codon without changing theencoded amino acid sequence; andd) adjusting the number of A and/or U nucleotides in the RNA sequence toa pre-set number of A and/or U nucleotides by replacing at least oneoriginal codon with an alternative codon, wherein the alternative codonencodes the same amino acid as the original codon and is furthercharacterized in a higher content of A and/or U nucleotides.

In preferred embodiments, step b) of the method further comprisesdetermining the number of G and/or C nucleotides in the original RNAsequence.

Preferably, the method for providing an adapted RNA according to theinvention further comprises a step e) which comprises producing the RNA.Preferably, the RNA is produced by any suitable method known in the artor by a method as described herein under the section ‘RNA production’.More preferably, the method involves a step of in vitro transcription,preferably as described herein. In a particularly preferred embodiment,the method involves in vitro transcription and a step of purifying theobtained RNA in a chromatographic process, preferably an HPLC process,more preferably an RP-HPLC process, preferably as described herein.

In the following, the concept of the inventive method for providing anadapted RNA is further illustrated by describing a preferred embodiment(see also Example 6 herein). The basic principle of sequence adaptation,particularly of AU adaptation by changing the codon usage, isexemplarily also illustrated in FIG. 2.

An automated in sllico method (algorithm) may be used to set the numberof any nucleotide in an RNA sequence to a defined value, withoutaltering the amino acid sequence (step d) of the method). In the contextof the invention, the automated in silico method may used for sequenceadaptation, in particular adaptation of the number of A and/or Unucleotides (AU count)) of RNA sequences in order to allow harmonizationof RNA mixtures for HPLC co-analysis and/or HPLC co-purification.

Table 1 below summarizes the codon changes (for each genetically encodedamino acid) in the coding sequence that may be applied to increase thenumber of A and/or U nucleotides of an RNA sequence, without changingthe encoded amino acid sequence. Table 2 summarizes the codon changes(for each genetically encoded amino acid) in the coding sequence thatmay be applied to decrease the number of A and/or U nucleotides of anRNA sequence, without changing the encoded amino acid sequence.

TABLE 1 Codon changes that allow for increase in the number of A and/orU nucleotides Amino Codon for Change in acid codon AU increase AU countAla GCG GCA/GCU +1 Ala GCA n.a. Ala GCU n.a. Ala GCC GCA/GCU +1 Cys UGUn.a. Cys UGC UGU +1 Asp GAU n.a. Asp GAC GAU +1 Glu GAG GAA +1 Glu GAAn.a. Phe UUU n.a. Phe UUC UUU +1 Gly GGG GGA/GGU +1 Gly GGA n.a. Gly GGUn.a. Gly GGC GGA/GGU +1 His CAU n.a. His CAC CAU +1 Ile AUA n.a. Ile AUUn.a. Ile AUC AUA/AUU +1 Lys AAG AAA +1 Lys AAA n.a. Leu UUG UUA +1 LeuUUA n.a. Leu CUG UUG/CUA/CUU +1 Leu CUG UUA +2 Leu CUA UUA +1 Leu CUUUUA +1 Leu CUC UUG/CUA/CUU +1 Leu CUC UUA +2 Met AUG n.a. Asn AAU n.a.Asn AAC n.a. Pro CCG CCU/CCA +1 Pro CCA n.a. Pro CCU n.a. Pro CCCCCU/CCA +1 Gln CAG CAA +1 Gln CAA n.a. Arg AGG AGA +1 Arg AGA n.a. ArgCGG CGU/CGA +1 Arg CGG AGA +2 Arg CGA AGA +1 Arg CGU AGA +1 Arg CGCCGU/CGA +1 Arg CGC AGA +2 Ser AGU n.a. Ser AGC AGU/UCA/UCU +1 Ser UCGAGU/UCA/UCU +1 Ser UCA n.a. Ser UCU n.a. Ser UCC AGU/UCA/UCU +1 Thr ACGACA/ACU +1 Thr ACA n.a. Thr ACU n.a. Thr ACC ACA/ACU +1 Val GUG GUA/GUU+1 Val GUA n.a. Val GUU n.a. Val GUC GUA/GUU +1 Trp UGG n.a. Tyr UAUn.a. Tyr UAC UAU +1 Stop UGA* UAA +1 Stop UAG UAA +1 Stop UM n.a. — n.a.= not applicable

TABLE 2 Codon changes that allow for decrease in the number of A and/orU nucleotides Amino Codon for Change in acid codon AU increase AU countAla GCG n.a. Ala GCA GCG/GCC −1 Ala GCU GCG/GCC −1 Ala GCC n.a. Cys UGUUGC −1 Cys UGC n.a. Asp GAU GAC −1 Asp GAC n.a. Glu GAG n.a. Glu GAA GAG−1 Phe UUU UUC −1 Phe UUC n.a. Gly GGG n.a. Gly GGA GGG/GGC −1 Gly GGUGGG/GGC −1 Gly GGC n.a. His CAU CAC −1 His CAC n.a. Ile AUA AUC −1 IleAUU AUC −1 Ile AUC n.a. Lys AAG n.a. Lys AAA AAG −1 Leu UUG CUG/CUC −1Leu UUA UUG/CUA/CUU −1 Leu UUA CUG/CUC −2 Leu CUG n.a. Leu CUA CUG/CUC−1 Leu CUU CUG/CUC −1 Leu CUC n.a. Met AUG n.a. Asn AAU n.a. Asn AACn.a. Pro CCG CCC −1 Pro CCA CCG −1 Pro CCU CCG −1 Pro CCA CCC −2 Pro CCUCCC −2 Pro CCC n.a. Gln CAG n.a. Gln CAA CAG −1 Arg AGG CGC/CGG −1 ArgAGA AGG/CGA/CGU −1 Arg AGA CGC/CGG −2 Arg CGG n.a. Arg CGA CGC/CGG −1Arg CGU CGC/CGG −1 Arg CGC n.a. Ser AGU n.a. Ser AGC n.a. Ser UCG n.a.Ser UCA UCG/AGC −1 Ser UCU UCG/AGC −1 Ser UCC n.a. Thr ACG n.a. Thr ACAACG/ACC −1 Thr ACU ACG/ACC −1 Thr ACC n.a. Val GUG n.a. Val GUA GUG/GUC−1 Val GUU GUG/GUC −1 Val GUC n.a. Trp UGG n.a. Tyr UAU UAC −1 Tyr UACn.a. Stop UGA n.a. Stop UAG n.a. Stop UAA UGA/UAG −1 n.a. = notapplicable

In the initial phase of the method, a matrix for each codon comprised inthe RNA sequence to be adapted is preferably created, identifyingpossible codon exchanges, which do not result in a change of the encodedamino acid (herein referred to as “exchange matrix”). An exemplary“exchange matrix” is shown in Formula (I).

$\begin{matrix}{\left. \begin{matrix}A & 1 \\C & 1 \\G & 1 \\T & 1 \\* & 4\end{matrix} \right\}{CGA}} & {{Formula}\mspace{14mu}(I)}\end{matrix}$

Formula (I) shows that for codon “CGA” a change to an alternative codonoffers the option of increasing the number of A nucleotides by 1 (e.g.:CGA->AGA), offers the option of increasing the number of C nucleotidesby 1 (e.g. CGA->CGC), offers the option of increasing the number of Gnucleotides by 1 (e.g. CGA->CGG), and offers the option of increasingthe number of T nucleotides by 1 (e.g. CGA->CGT).

Exchange matrices are preferably generated for each individual codon inthe RNA sequence to be adapted. Using said exchange matrices, thepotential maximum number of the respective nucleotides (A and T(U)count, respectively) in each codon may be determined (without changingthe amino acid sequence). Accordingly, all codons of the sequence, whichis to be adapted, are analyzed with respect to potential codon exchangesby step-wise iteration, wherein in each iteration step the correspondingcodon is analysed using the respective exchange matrix (as outlinedabove) for potential nucleotide changes. For example, if no changes aretheoretically possible in the respective codon, e.g. as in the case of“ATG” or “TGG”, the corresponding exchange matrix as exemplarily shownin Formula (II) was used (* of exchange matrix=0).

$\begin{matrix}{\left. \begin{matrix}A & 0 \\C & 0 \\G & 0 \\T & 0 \\* & 0\end{matrix} \right\}{ATG}} & {{Formula}\mspace{14mu}({II})}\end{matrix}$

Formula (II) shows that for codon “ATG” a change to an alternative codonoffers no option of increasing the number of A nucleotides, Cnucleotides, G nucleotides or T nucleotides (as there are no alternativecodons for ATG (Met)).

In cases where exchanges according to the respective exchange matrix(*>0) are theoretically possible, the codon is further analysed if thechange can be implemented under the premise that e.g. only codons thatoffer the option of increasing the number of A and/or T(U) nucleotidesare adapted. Therefore the intersection between the target nucleotides(e.g. A and/or T(U)) and the nucleotides that potentially generate apositive result (that is, A and/or T(U) change; see e.g. Formula (I)) inthe current exchange matrix are constructed. As a result, each codon maybe categorized and grouped into one of three categories:

Category 1 (Category “Favourable”):

Potential codon exchanges allowing an increase in only one targetnucleotide (in the present example A or T(U)). For example, the codon“GAC” (Asp) can be changed to “GAU” (Asp) in order to increase thenumber of A and T(U) nucleotides. No further analysis is required sincethat modification does not have any further impact (besides the onementioned above) on the number of A and T(U) nucleotides.

Category 2 (Category “Possible”):

Potential codon exchanges allowing the increase in both targetnucleotides (in the present example A and T(U)). For example, codon“GCA” can be changed to “GCU”, which would increase the T(U) count butat the same time decrease the A count. Accordingly, further analysiswould be required with respect to codons belonging to this the categoryin order to decide, whether the number of one of the two targetnucleotides (T(U)) in this example should be increased at the expense ofa reduction of the number of the other target nucleotide (A).

Category 3 (Category “Impossible”):

Codons in the RNA sequence, for which no alternative codons exist (*=0).Examples for this category 3 are ATG (Met; start codon) or “UGG” (Trp).

All codons of the original sequence are preferably categorized in thatmanner. It may then preferably be calculated how many potentialnucleotide exchanges have been identified for all target nucleotides (A,T(U)).

In preferred embodiments, the method comprises adapting the RNA sequenceusing only codons from category 1 as defined herein. In someembodiments, codons from category 2 may also be used (for example, assoon as all codons from category 1 have been used), thus offeringadditional adaptation possibilities for A and T(U) counts. If category 2is required in order to achieve the desired nucleotide counts,calculation of the following ratio may be used for identifying theexchange nucleotide (nucleotide A or T(U)):

$\frac{c_{i}}{x_{i} - p_{i}}$

wherein i represents the corresponding target, c_(i) is the count ofpossible adaptation positions of i in category 2, x_(i) is the desiredthreshold for i, p_(i) the count for the already changed identifiedadaptation positions. All calculated ratios are preferably ranked andstarting from lowest to highest ratio, the changes from category 2 areapplied, until the desired threshold has been reached or until all thepossible exchanges from category 2 have been performed. This procedureis preferably carried out iteratively for any RNA sequence, where thetarget nucleotide count, e.g. the target number of A and/or Unucleotides, cannot be achieved by only using exchanges according tocategory 1.

In cases where the desired target nucleotide count cannot be achieved(as all alternative codons from category 1 and 2 have been used, whichmeans that no further changes are possible), an adapted sequence ispreferably generated that is matching the target nucleotide count asclose as possible.

According to some embodiments of the method, one of the followingimprovements may be implemented therein:

-   1. Besides the basic equal distribution, which is used in the    embodiment described above and which is based on the exchange    possibilities, other distribution models may also be envisaged, such    as normal distributed, first occurrences distribution, last    occurrences distribution or random-based distribution.    Alternatively, the mean of the possible changes or median of the    possible changes may be determined and all exchanges may be arranged    around these values.-   2. The exchange matrix may further contain additional information    about the codon for the target sequence (e.g. codon usage etc.).    This creates a further criteria for the question of whether a codon    exchange is desirable or not, facilitating adaptation to a specific    codon usage or a different nucleotide ratio in the target sequence.-   3. A third category may further be implemented by sequences or    motifs, which should be avoided by an exchange (e.g. a recognition    motif of a restriction enzyme, promotor sequences or sequences    building not desired secondary structures, etc.).-   4. Automated binning of input sequences may be performed, based on    their length and the occurrence of the desired target nucleotides in    order to identify optimal nucleotide counts for A and/or U    adaptation.

Any one of the steps or combination of steps described with respect tothe method for providing an adapted RNA according to the invention maypreferably be applied to the other methods described herein, inparticular to the method for modifying the retention time of an RNA on achromatographic column, to the method for purifying at least one RNAspecies from a mixture of at least two RNA species, to the method forco-purifying at least two RNA species from a mixture comprising at leasttwo RNA species, or to the method for harmonizing the numbers of Aand/or U nucleotides in the sequences of at least two RNA species.

Composition and Vaccine

In a further aspect, the present invention provides a compositioncomprising at least one adapted RNA sequence as described herein and,optionally, a pharmaceutically acceptable carrier. The inventivecomposition comprising the at least one adapted RNA as described hereinis preferably a pharmaceutical composition or a vaccine as describedherein. According to a preferred embodiment, the inventive compositionis a polyvalent vaccine, preferably as described herein.

In preferred embodiments, the composition according to the inventioncomprises an RNA obtainable by a method as described herein, inparticular by the method for modifying the retention time of an RNA on achromatographic column, the method for purifying at least one RNAspecies from a mixture of at least two RNA species, the method forco-purifying at least two RNA species from a mixture comprising at leasttwo RNA species, the method for harmonizing the numbers of A and/or Unucleotides in the sequences of at least two RNA species, or the methodfor providing an adapted RNA as described herein.

According to a preferred embodiment, the composition according to theinvention comprises at least two RNA species, wherein the sequence of atleast one RNA species is adapted by altering the number of A and/or Unucleotides in the sequence of the at least one RNA species with respectto the number of A and/or U nucleotides in the original RNA sequence.More preferably, the composition comprises at least two RNA species,wherein at least one RNA species is an adapted RNA as obtained by anyone of the methods according to the invention, preferably by the methodfor modifying the retention time of an RNA on a chromatographic column,the method for purifying at least one RNA species from a mixture of atleast two RNA species, the method for co-purifying at least two RNAspecies from a mixture comprising at least two RNA species, the methodfor harmonizing the numbers of A and/or U nucleotides in the sequencesof at least two RNA species, or the method for providing an adapted RNAas described herein. More preferably, the numbers of A and/or Unucleotides in the sequences of the at least two RNA species comprisedin the inventive composition are harmonized as described herein.

In preferred embodiments, the composition according to the inventioncomprises at least two RNA species, e.g., at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, or at least 10RNA species, wherein the sequence of at least one RNA species is adaptedby altering the number of A and/or U nucleotides in the sequence of theat least one RNA species with respect to the number of A and/or Unucleotides in the original RNA sequence, wherein the numbers of Aand/or U nucleotides in the two (or more) RNA species preferably differfrom each other by not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150. Inpreferred embodiments, the numbers of A and/or U nucleotides in the RNAspecies preferably differ from each other by not more than 2, morepreferably by not more than 3, even more preferably by not more than 4,even more preferably by not more than 5, most preferably by not morethan 6. In a further preferred embodiment, the numbers of A and/or Unucleotides in the RNA species differ from each other by not more than10, preferably by not more than 20, more preferably by not more than 30,even more preferably by not more than 40, most preferably by not morethan 50. It is particularly preferred, that the numbers of A and/or Unucleotides in the two or more RNA species are identical.

Preferably, the inventive composition comprises or consists of at leastone adapted RNA (species) as described herein and a pharmaceuticallyacceptable carrier. The expression “pharmaceutically acceptable carrier”as used herein preferably includes the liquid or non-liquid basis of theinventive composition, which is preferably a pharmaceutical compositionor a vaccine. If the inventive composition is provided in liquid form,the carrier will preferably be water, typically pyrogen-free water;isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrateetc. buffered solutions. Water or preferably a buffer, more preferablyan aqueous buffer, may be used, containing a sodium salt, preferably atleast 50 mM of a sodium salt, a calcium salt, preferably at least 0.01mM of a calcium salt, and optionally a potassium salt, preferably atleast 3 mM of a potassium salt. According to a preferred embodiment, thesodium, calcium and, optionally, potassium salts may occur in the formof their halogenides, e.g. chlorides, iodides, or bromides, in the formof their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.Without being limited thereto, examples of sodium salts include e.g.NaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, examples of the optionalpotassium salts include e.g. KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, andexamples of calcium salts include e.g. CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄,Ca(OH)₂. Furthermore, organic anions of the aforementioned cations maybe contained in the buffer.

Furthermore, one or more compatible solid or liquid fillers or diluentsor encapsulating compounds may be used as well, which are suitable foradministration to a person. The term “compatible” as used herein meansthat the constituents of the inventive composition are capable of beingmixed with the at least one (adapted) RNA (species) of the composition,in such a manner that no interaction occurs, which would substantiallyreduce the biological activity or the pharmaceutical effectiveness ofthe inventive composition under typical use conditions. Pharmaceuticallyacceptable carriers, fillers and diluents must, of course, havesufficiently high purity and sufficiently low toxicity to make themsuitable for administration to a person to be treated. Some examples ofcompounds which can be used as pharmaceutically acceptable carriers,fillers or constituents thereof are sugars, such as, for example,lactose, glucose, trehalose and sucrose; starches, such as, for example,corn starch or potato starch; dextrose; cellulose and its derivatives,such as, for example, sodium carboxymethylcellulose, ethylcellulose,cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solidglidants, such as, for example, stearic acid, magnesium stearate;calcium sulfate; vegetable oils, such as, for example, groundnut oil,cottonseed oil, sesame oil, olive oil, corn oil and oil from Theobroma;polyols, such as, for example, polypropylene glycol, glycerol, sorbitol,mannitol and polyethylene glycol; alginic acid.

Further additives, which may be included in the composition areemulsifiers, such as, for example, Tween; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

In a preferred embodiment the (adapted) RNA as defined herein, comprisedin the composition, the pharmaceutical composition, the vaccine asdefined herein, is complexed or at least partially complexed orassociated with one or more cationic or polycationic compound preferablywith cationic or polycationic polymer, cationic or polycationicpolysaccharide, cationic or polycationic lipid, cationic or polycationicprotein, cationic or polycationic peptide, or any combinations thereof.

The term “cationic or polycationic compound” as used herein will berecognized and understood by the person of ordinary skill in the art,and are for example intended to refer to a charged molecule, which ispositively charged at a pH value ranging from about 1 to 9, at a pHvalue ranging from about 3 to 8, at a pH value ranging from about 4 to8, at a pH value ranging from about 5 to 8, more preferably at a pHvalue ranging from about 6 to 8, even more preferably at a pH valueranging from about 7 to 8, most preferably at a physiological pH, e.g.ranging from about 7.2 to about 7.5. Accordingly, a cationic component,e.g. a cationic peptide, cationic protein, cationic polymer, cationicpolysaccharide, cationic lipid may be any positively charged compound orpolymer which is positively charged under physiological conditions. A“cationic or polycationic peptide or protein” may contain at least onepositively charged amino acid, or more than one positively charged aminoacid, e.g. selected from Arg, His, Lys or Orn. Accordingly,“polycationic” components are also within the scope exhibiting more thanone positive charge under the given conditions.

Cationic or polycationic compounds, being particularly preferred in thiscontext may be selected from the following list of cationic orpolycationic peptides or proteins of fragments thereof: protamine,nucleoline, spermine or spermidine, or other cationic peptides orproteins, such as poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs), PpT620, prolin-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides,pAntp, pIsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB,SynB(1), pVEC, hCT-derived peptides, SAP, or histones. More preferably,the nucleic acid as defined herein, preferably the mRNA as definedherein, is complexed with one or more polycations, preferably withprotamine or oligofectamine, most preferably with protamine. In thiscontext protamine is particularly preferred.

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene etc.; cationic lipids,e.g. DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9,oligofectamine; or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as beta-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP etc., modified acrylates, suchas pDMAEMA etc., modified amidoamines such as pAMAM etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI, poly(propyleneimine), etc., polyallylamine,sugar backbone based polymers, such as cyclodextrin based polymers,dextran based polymers, etc., silan backbone based polymers, such asPMOXA-PDMS copolymers, etc., blockpolymers consisting of a combinationof one or more cationic blocks (e.g. selected from a cationic polymer asmentioned above) and of one or more hydrophilic or hydrophobic blocks(e.g. polyethyleneglycole); etc.

In embodiments, the composition or vaccine comprises at least one(adapted)RNA as described herein, which is complexed with one or morepolycationic compounds and/or a polymeric carrier, and at least one freeRNA, wherein the at least one complexed RNA is preferably identical tothe at least one RNA according to the present invention.

The term “polymeric carrier” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and are forexample intended to refer to a compound that facilitates transportand/or complexation of another compound (cargo). A polymeric carrier istypically a carrier that is formed of a polymer. A polymeric carrier maybe associated to its cargo (nucleic acid, RNA) by covalent ornon-covalent interaction

In this context it is particularly preferred that the at least one RNAof the inventive composition is complexed at least partially with acationic or polycationic compound and/or a polymeric carrier, preferablycationic proteins or peptides. In this context, the disclosure ofWO2010/037539 and WO2012/113513 is incorporated herewith by reference.Partially means that only a part of the artificial nucleic acid iscomplexed with a cationic compound and that the rest of the artificialnucleic acid is (comprised in the inventive pharmaceutical composition,immunogenic composition) in uncomplexed form (“free”).

Further preferred cationic or polycationic proteins or peptides may bederived from formula (Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x of the patentapplication WO2009/030481, the disclosure of WO2009/030481 relatingthereto incorporated herewith by reference.

According to a preferred embodiment, the composition of the presentinvention comprises the RNA as defined herein, and a polymeric carrier.A polymeric carrier used according to the invention might be a polymericcarrier formed by disulfide-crosslinked cationic components. Thedisulfide-crosslinked cationic components may be the same or differentfrom each other. The polymeric carrier can also contain furthercomponents. It is also particularly preferred that the polymeric carrierused according to the present invention comprises mixtures of cationicpeptides, proteins or polymers and optionally further components asdefined herein, which are crosslinked by disulfide bonds as describedherein.

In this context, polymeric carriers according to formula{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa′)x(Cys)y} and formulaCys,{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys₂ of the patent applicationWO2012/013326 are preferred, the disclosure of WO2012/013326 relatingthereto incorporated herewith by reference.

In a further particular embodiment, the polymeric carrier which may beused to complex the RNA as defined herein or any further nucleic acidcomprised in the (pharmaceutical) composition or vaccine according tothe invention may be derived from a polymeric carrier molecule accordingformula (L-P¹—S—[S—P²—S]_(n)—S—P³-L) of the patent applicationWO2011/026641, the disclosure of WO2011/026641 relating theretoincorporated herewith by reference.

In other embodiments, the composition, which is preferably acomposition, a pharmaceutical composition or a vaccine, comprises atleast one artificial nucleic acid as described herein, wherein the atleast one artificial nucleic acid is complexed or associated withpolymeric carriers and, optionally, with at least one lipid component asdescribed in the PCT applications PCT/EP2016/06322, PCT/EP2016/063227,PCT/EP2016/063229, PCT/EP2016/063226. In this context, the disclosuresof PCT/EP2016/06322, PCT/EP2016/063227, PCT/EP2016/063229,PCT/EP2016/063226 is herewith incorporated by reference.

In preferred embodiments, the polymeric carrier compound is formed by,or comprises or consists of the peptide elements CysArg12Cys or CysArg12or TrpArg12Cys. In particularly preferred embodiments, the polymericcarrier compound consists of a (R₁₂C)—(R₁₂C) dimer, a (WR₁₂C)—(WR₁₂C)dimer, or a (CR₁₂)—(CR₁₂C)—(CR₁₂) trimer, wherein the individual peptideelements in the dimer (e.g. (WR12C)), or the trimer (e.g. (CR12)), areconnected via —SH groups.

In embodiments, where the complexed (adapted) RNA is complexed withcationic or polycationic peptides or proteins as the carrier compound,the nitrogen/phosphate ratio of the complexed RNA ranges from about 0.1to about 20, or from about 0.2 to about 15, or from about 2 to about 15,or from about 2 to about 12, wherein the N/P ratio is defined as themole ratio of the nitrogen atoms of the basic groups of the cationicpeptide or polymer to the phosphate groups of the nucleic acid,preferably the RNA.

Accordingly, the composition as defined herein, comprising at least oneRNA as defined herein, wherein the N/P ratio of the at least oneartificial nucleic acid, preferably the RNA as defined herein, to theone or more cationic or polycationic compound as defined herein,preferably protamine, is in the range of about 0.1 to 20, including arange of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and ofabout 0.7 to 1.5.

Suitably, the at least one RNA as defined herein, is complexed with oneor more cationic or polycationic compounds as defined herein, in aweight ratio selected from a range of about 6:1 (w/w) to about 0.25:1(w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), evenmore preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)to about 2:1 (w/w) of nucleic acid to cationic or polycationic compoundand/or with a polymeric carrier; or optionally in a nitrogen/phosphateratio of nucleic acid to cationic or polycationic component and/orpolymeric carrier in the range of about 0.1-10, preferably in a range ofabout 0.3-4 or 0.3-1, and most preferably in a range of about 0.5-1 or0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9.

In this context it is particularly preferred that the at least one RNAas defined herein is complexed with protamine.

Suitably, the complexed RNA is complexed with protamine by addition ofprotamine-trehalose solution to the RNA sample at an RNA to protamineweight to weight ratio (w/w) of 2:1.

In preferred embodiments, the composition, the pharmaceuticalcomposition, the vaccine as defined herein comprises the at least oneRNA as defined herein which is complexed with one or more cationic orpolycationic compounds (e.g. protamine), and at least one free RNA.

In preferred embodiments, the at least one complexed RNA (e.g. protaminecomplexed RNA) is identical to the at least one free RNA.

Preferably, the molar ratio of the RNA of the adjuvant component (e.g.protamine-complexed RNA) to the free nucleic acid, particularly the freeRNA may be selected from a molar ratio of about 0.001:1 to about1:0.001, including a ratio of about 1:1.

Preferably the ratio of complexed RNA of the adjuvant component (e.g.protamine-complexed RNA), to free nucleic acid, particularly the freeRNA, may be selected from a range of about 5:1 (w/w) to about 1:10(w/w), more preferably from a range of about 4:1 (w/w) to about 1:8(w/w), even more preferably from a range of about 3:1 (w/w) to about 1:5(w/w) or 1:3 (w/w), wherein the ratio is most preferably about 1:1(w/w).

With respect to a composition comprising an adjuvant component (e.g.protamine-complexed RNA) and a free RNA component as defined herein, thedisclosure of WO2009/144230 is incorporated herewith by reference. Sucha composition comprising an adjuvant component as defined herein (e.g.protamine complexed RNA) and a free RNA component as defined herein maybe generated using means and methods as disclosed in WO2016/165825.

In preferred embodiments, the composition, which is preferably apharmaceutical composition, a vaccine, comprises at least one(adapted)RNA as described herein, wherein the at least one RNA iscomplexed or associated with one or more lipids (e.g. cationic lipidsand/or neutral lipids), thereby forming liposomes, lipid nanoparticles(LNPs), lipoplexes, and/or nanoliposomes.

In the context of the present invention, the term “lipid nanoparticle”,also referred to as “LNP”, is not restricted to any particularmorphology, and include any morphology generated when a cationic lipidand optionally one or more further lipids are combined, e.g. in anaqueous environment and/or in the presence of an RNA. For example, aliposome, a lipid complex, a lipoplex and the like are within the scopeof a lipid nanoparticle (LNP).

LNPs typically comprise a cationic lipid and one or more excipientselected from neutral lipids, charged lipids, steroids and polymerconjugated lipids (e.g. PEGylated lipid). The RNA may be encapsulated inthe lipid portion of the LNP or an aqueous space enveloped by some orthe entire lipid portion of the LNP. The RNA or a portion thereof mayalso be associated and complexed with the LNP. An LNP may comprise anylipid capable of forming a particle to which the nucleic acids areattached, or in which the one or more nucleic acids are encapsulated.Preferably, the LNP comprising nucleic acids comprises one or morecationic lipids, and one or more stabilizing lipids. Stabilizing lipidsinclude neutral lipids and PEGylated lipids.

In one embodiment, the LNP consists essentially of (i) at least onecationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol;and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, in a molar ratio ofabout 20-60% cationic lipid:5-25% neutral lipid:25-55% sterol; 0.5-15%PEG-lipid.

In that context, a preferred sterol is cholesterol. The sterol can beabout 10 mol % to about 60 mol % or about 25 mol to about 40 mol % ofthe lipid particle. In one embodiment, the sterol is about 10, 15, 20,25, 30, 35, 40, 45, 50, 55, or about 60 mol % of the total lipid presentin the lipid particle. In another embodiment, the LNPs include fromabout 5% to about 50% on a molar basis of the sterol, e.g., about 15% toabout 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%,about 35%, about 34.4%, about 31.5% or about 31% on a molar basis (basedupon 100% total moles of lipid in the lipid nanoparticle).

The cationic lipid of an LNP may be cationisable, i.e. it becomesprotonated as the pH is lowered below the pK of the ionizable group ofthe lipid, but is progressively more neutral at higher pH values. At pHvalues below the pK, the lipid is then able to associate with negativelycharged nucleic acids. In certain embodiments, the cationic lipidcomprises a zwitterionic lipid that assumes a positive charge on pHdecrease.

The LNP may comprise any further cationic or cationisable lipid, i.e.any of a number of lipid species which carry a net positive charge at aselective pH, such as physiological pH.

Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);3-(N—(N′,N′dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS),1,2-dioleoyl-3-dimethylammonium propane (DODAP),N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), andN-(1,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE).

Additionally, a number of commercial preparations of cationic lipids areavailable which can be used in the present invention. These include, forexample, LIPOFECTIN® (commercially available cationic liposomescomprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), fromGIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially availablecationic liposomes comprisingN-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.). The following lipids are cationic and have apositive charge at below physiological pH: DODAP, DODMA, DMDMA,1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

The further cationic lipid may also be an amino lipid. Representativeamino lipids include, but are not limited to,1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanediol (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA);dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3(US20100324120).

Other suitable (cationic) lipids are disclosed in WO2009/086558,WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537,WO2010/129709, WO2011/153493, US2011/0256175, US2012/0128760,US2012/0027803, and U.S. Pat. No. 8,158,601. In that context, thedisclosures of WO2009/086558, WO2009/127060, WO2010/048536,WO2010/054406, WO2010/088537, WO2010/129709, WO2011/153493,US2011/0256175, US2012/0128760, US2012/0027803, and U.S. Pat. No.8,158,601 are incorporated herewith by reference.

In a particularly preferred embodiment the LNP comprises a cationiclipid with the formula (III) according to the patent applicationPCT/EP2016/075929. In this context, the disclosure of PCT/EP2016/075929relating to cationic lipids is incorporated herewith by reference.

The amount of the permanently cationic lipid or lipidoid may be selectedtaking the amount of the nucleic acid cargo into account. In oneembodiment, these amounts are selected such as to result in an N/P ratioof the nanoparticle(s) or of the composition in the range from about 0.1to about 20. In this context, the N/P ratio is defined as the mole ratioof the nitrogen atoms (“N”) of the basic nitrogen-containing groups ofthe lipid or lipidoid to the phosphate groups (“P”) of the RNA which isused as cargo. The N/P ratio may be calculated on the basis that, forexample, 1 μg RNA typically contains about 3 nmol phosphate residues,provided that the RNA exhibits a statistical distribution of bases. The“N”-value of the lipid or lipidoid may be calculated on the basis of itsmolecular weight and the relative content of permanently cationic and—ifpresent—cationisable groups.

In certain embodiments, the LNP comprises one or more additional lipidswhich stabilize the formation of particles during their formation.

Suitable stabilizing lipids include neutral lipids and anionic lipids.The term “neutral lipid” refers to any one of a number of lipid speciesthat exist in either an uncharged or neutral zwitterionic form atphysiological pH. Representative neutral lipids includediacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides,sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

Exemplary neutral lipids include, for example,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE). In one embodiment, the neutral lipid is1,2-distearoyl-sn-glycero-3phosphocholine (DSPC).

In some embodiments, the LNPs comprise a neutral lipid selected fromDSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, themolar ratio of the cationic lipid to the neutral lipid ranges from about2:1 to about 8:1.

LNP in vivo characteristics and behavior can be modified by addition ofa hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to theLNP surface to confer steric stabilization. Furthermore, LNPs can beused for specific targeting by attaching ligands (e.g. antibodies,peptides, and carbohydrates) to its surface or to the terminal end ofthe attached PEG chains (e.g. via PEGylated lipids).

In some embodiments, the LNPs comprise a polymer conjugated lipid. Theterm “polymer conjugated lipid” refers to a molecule comprising both alipid portion and a polymer portion. An example of a polymer conjugatedlipid is a PEGylated lipid. The term “PEGylated lipid” refers to amolecule comprising both a lipid portion and a polyethylene glycolportion. PEGylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG)and the like.

In certain embodiments, the LNP comprises an additional,stabilizing-lipid which is a polyethylene glycol-lipid (PEGylatedlipid). Suitable polyethylene glycol-lipids include PEG-modifiedphosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modifiedceramides (e.g. PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols.Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA,and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid isN-[(methoxy poly(ethyleneglycol)₂₀₀₀)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). Inone embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In otherembodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) suchas 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG),a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asw-methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl) carbamate. Invarious embodiments, the molar ratio of the cationic lipid to thePEGylated lipid ranges from about 100:1 to about 25:1.

The total amount of nucleic acid, particularly the RNA in the lipidnanoparticles varies and may be defined depending on the e.g. RNA tototal lipid w/w ratio. In one embodiment of the invention the RNA tototal lipid ratio is less than 0.06 w/w, preferably between 0.03 w/w and0.04 w/w.

The composition, pharmaceutical composition, or vaccine comprising atleast one (adapted) RNA according to the invention may be provided inliquid and or in dry (e.g. lyophylized) form. In a preferred embodiment,the RNA according to the invention or the composition is provided inlyophilized form. The RNA and the composition thus provide a possibilityto store (irrespective of the ambient temperature and also withoutcooling) an RNA and a composition suitable for vaccination against, forexample, an infectious disease, such as an infection with influenzavirus or norovirus. Preferably, the at least one lyophilized RNA orcomposition is reconstituted in a suitable buffer, advantageously basedon an aqueous carrier, e.g. Ringer-Lactate solution, prior to use, suchas administration to a subject.

In a preferred embodiment, the composition is a pharmaceuticalcomposition or a vaccine, which typically comprises a safe and effectiveamount of at least one (adapted) RNA as defined herein. As used herein,“safe and effective amount” means an amount of the RNA of thecomposition or vaccine as defined above, that is sufficient tosignificantly induce an immune response against, for example, aninfectious disease as described herein, such as an infection withinfluenza virus. At the same time, however, a “safe and effectiveamount” is small enough to avoid serious side effects that is to say topermit a sensible relationship between advantage and risk. Thedetermination of these limits typically lies within the scope ofsensible medical judgment. In relation to the vaccine or composition,the expression “safe and effective amount” preferably means an amount ofthe RNA that is suitable for stimulating the adaptive immune system insuch a manner that no excessive or damaging immune reactions areachieved but, preferably, also no such immune reactions below ameasurable level. Such a “safe and effective amount” of the RNA of thecomposition or vaccine as defined above may furthermore be selected, forexample in dependence of the type of RNA, e.g. monocistronic, bi- oreven multicistronic mRNA, since a bi- or even multicistronic mRNA maylead to a significantly higher expression of the encoded polypeptide(s)than use of an equal amount of a monocistronic mRNA. A “safe andeffective amount” of the RNA of the composition or vaccine as definedabove may furthermore vary in connection with the particular objectiveof the treatment and also with the age and physical condition of thepatient to be treated, and similar factors, within the knowledge andexperience of the accompanying doctor. The vaccine or compositionaccording to the invention can be used according to the invention forhuman and also for veterinary medical purposes, as a pharmaceuticalcomposition or as a vaccine.

In a preferred embodiment, the RNA of the composition, vaccine or kit ofparts according to the invention is provided in lyophilized form.Preferably, the lyophilized RNA is reconstituted in a suitable buffer,advantageously based on an aqueous carrier, prior to administration,e.g. Ringer-Lactate solution, which is preferred, Ringer solution, aphosphate buffer solution.

According to a preferred embodiment, the buffer suitable for injectionmay be used as a carrier in the inventive vaccine or composition or forresuspending the inventive vaccine or the inventive composition. Such abuffer suitable for injection may be, for example, the liquid ornon-liquid basis/carrier as described herein. Ringer-Lactate solution isparticularly preferred as a liquid basis.

The choice of a pharmaceutically acceptable carrier is determined, inprinciple, by the manner, in which the vaccine or the composition isadministered. The vaccine or composition can be administered, forexample, systemically or locally. Routes for systemic administration ingeneral include, for example, transdermal, oral, parenteral routes,including subcutaneous, intravenous, intramuscular, intraarterial,intradermal and intraperitoneal injections and/or intranasaladministration routes. Routes for local administration in generalinclude, for example, topical administration routes but alsointradermal, transdermal, subcutaneous, or intramuscular injections orintralesional, intracranial, intrapulmonal, intracardial, and sublingualinjections. More preferably, the vaccine or the composition may beadministered by an intradermal, subcutaneous, or intramuscular route,preferably by injection, which may be needle-free and/or needleinjection. Compositions/vaccines are therefore preferably formulated inliquid or solid form. The suitable amount of the vaccine or compositionto be administered can be determined by routine experiments with animalmodels. Such models include, without implying any limitation, rabbit,sheep, mouse, rat, dog and non-human primate models. Preferred unit doseforms for injection include sterile solutions of water, physiologicalsaline or mixtures thereof. The pH of such solutions should be adjustedto about 7.4. Suitable carriers for injection include hydrogels, devicesfor controlled or delayed release, polylactic acid and collagenmatrices. Suitable pharmaceutically acceptable carriers for topicalapplication include those which are suitable for use in lotions, creams,gels and the like. If the vaccine is to be administered perorally,tablets, capsules and the like are the preferred unit dose form. Thepharmaceutically acceptable carriers for the preparation of unit doseforms which can be used for oral administration are well known in theprior art. The choice thereof will depend on secondary considerationssuch as taste, costs and storability, which are not critical for thepurposes of the present invention, and can be made without difficulty bya person skilled in the art.

According to another embodiment, the (pharmaceutical) composition or thevaccine may comprise an adjuvant. An adjuvant may be used, for example,in order to enhance the immunostimulatory properties of the vaccine orcomposition. In this context, an adjuvant may be understood as anycompound, which is suitable to support administration and delivery ofthe vaccine or composition according to the invention. Furthermore, suchan adjuvant may, without being bound thereto, initiate or increase animmune response of the innate immune system, i.e. a non-specific immuneresponse. In other words, when administered, the vaccine or compositionaccording to the invention typically initiates an adaptive immuneresponse due to the at least one antigenic peptide or protein containedin the vaccine or composition. Additionally, the vaccine or compositionaccording to the invention may generate an (supportive) innate immuneresponse due to addition of an adjuvant as defined herein to the vaccineor composition according to the invention.

Such an adjuvant may be selected from any adjuvant known to a skilledperson and suitable for the present case, i.e. supporting the inductionof an immune response in a mammal. Preferably, the adjuvant may beselected from the group consisting of, without being limited thereto,TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminiumhydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucansfrom algae; algammulin; aluminium hydroxide gel (alum); highlyprotein-adsorbing aluminium hydroxide gel; low viscosity aluminiumhydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%),Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE™(propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of:i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferon-gamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(beta-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acidand glycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA(polymethyl methacrylate); PODDS™ (proteinoid microspheres);polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylicacid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™(QS-21); Quil-A (Quil-A saponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5c]quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendaiproteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitantrioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85);squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin; microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine,IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPIanchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusionprotein, cdiGMP; and adjuvants suitable as antagonists including CGRPneuropeptide.

Further adjuvants that may be suitably used are also provided inWO2016/203025. With respect to suitable adjuvants that may be comprisedin order to enhance the immunostimulatory properties of the compositionaccording to the invention, the adjuvants according to claim 7 and/orclaim 17 of WO2016/203025, and the disclosure relating thereto, areincluded herewith by reference.

In certain embodiments, the composition, the pharmaceutical composition,the immunogenic composition as defined herein may comprise at least oneadjuvant, wherein the at least one adjuvant may be an nucleic acidadjuvant having the formula GlXmGn or nucleic acid adjuvant having theformula ClXmCn as disclosed in WO2008014979 and WO2009095226respectively, the disclosure relating thereto incorporated herein byreference.

The vaccine or composition can additionally contain one or moreauxiliary substances in order to further increase the immunogenicity. Asynergistic action of the RNA of the composition or vaccine as definedherein and of an auxiliary substance, which may be optionally beco-formulated (or separately formulated) with the vaccine or compositionas described above, is preferably achieved thereby. Depending on thevarious types of auxiliary substances, various mechanisms can come intoconsideration in this respect. For example, compounds that permit thematuration of dendritic cells (DCs), for example lipopolysaccharides,TNF-alpha or CD40 ligand form a first class of suitable auxiliarysubstances. In general, it is possible to use as auxiliary substance anyagent that influences the immune system in the manner of a “dangersignal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow animmune response produced by the immune-stimulating adjuvant according tothe invention to be enhanced and/or influenced in a targeted manner.Particularly preferred auxiliary substances are cytokines, such asmonokines, lymphokines, interleukins or chemokines, that—additional toinduction of the adaptive immune response by the encoded at least oneantigen—promote the innate immune response, such as IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha,IFN-beta, INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growthfactors, such as hGH. Preferably, such immunogenicity increasing agentsor compounds are provided separately (not co-formulated with theinventive vaccine or composition) and administered individually.

The vaccine or composition can also additionally contain any furthercompound, which is known to be immune-stimulating due to its bindingaffinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to the vaccine orcomposition in this context, may be CpG nucleic acids, in particularCpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be a single-strandedCpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), asingle-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA,more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). TheCpG nucleic acid preferably contains at least one or more (mitogenic)cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According to afirst preferred alternative, at least one CpG motif contained in thesesequences, that is to say the C (cytosine) and the G (guanine) of theCpG motif, is unmethylated. All further cytosines or guanines optionallycontained in these sequences can be either methylated or unmethylated.According to a further preferred alternative, however, the C (cytosine)and the G (guanine) of the CpG motif can also be present in methylatedform.

Preferably, the above compounds are formulated and administeredseparately from the above composition or vaccine (of the invention)containing the RNA according to the invention.

According to a preferred embodiment, the composition according to theinvention comprises at least two RNA species, wherein the sequence of atleast one RNA species is adapted by altering the number of A and/or Unucleotides in the sequence of the at least one RNA species with respectto the number of A and/or U nucleotides in the original RNA sequence,wherein at least one RNA species comprises at least one coding regionencoding at least one peptide or protein derived from a Norovirus, or afragment or a variant of said peptide or protein. Preferably, the atleast one adapted RNA in the inventive composition comprises at leastone coding region encoding a peptide or protein derived from aNorovirus, or a fragment or a variant of said peptide or protein. Morepreferably, the at least one coding region encodes a peptide or proteinderived from a Norovirus as described herein, or a fragment or variantthereof. In some embodiments, the composition of the present inventionis a Norovirus vaccine, more preferably a polyvalent Norovirus vaccine.

Suitable Norovirus antigens and antigenic peptides or proteins derivedfrom Norovirus are described in international patent applicationPCT/EP2017/060673, which is hereby incorporated by reference in itsentirety.

In preferred embodiments, the RNA (species) as used herein, preferablythe original RNA (species) that is to be adapted or the adapted RNA(species), comprises at least one coding region, which encodes a peptideor protein (derived from a Norovirus) comprising or consisting of anamino acid sequence according to any one of SEQ ID NOs: 1-4410 asdescribed in PCT/EP2017/060673, or a fragment or variant of any one ofthese amino acid sequences.

According to some embodiments, the RNA (species) used herein, preferablythe original RNA (species) that is to be adapted, comprises at least onecoding region comprising or consisting of a nucleic acid sequenceaccording to any one of SEQ ID NOs: 4411-39690, 39713-39746 as describedin PCT/EP2017/060673, or a fragment or variant of any one of thesenucleic acid sequences.

In a further preferred embodiment, the composition according to theinvention is a polyvalent Norovirus vaccine, which comprises at leasttwo RNA species, wherein the sequence of at least one RNA species isadapted by altering the number of A and/or U nucleotides in the sequenceof the at least one RNA species with respect to the number of A and/or Unucleotides in the original RNA sequence, wherein at least one RNAspecies comprises at least one coding region encoding at least onepeptide or protein derived from an Norovirus, or a fragment or a variantof said peptide or protein, and wherein at least one RNA speciescomprises at least one coding region encoding a different peptide orprotein derived from a Norovirus, or a fragment or a variant of saidpeptide or protein.

In a particularly preferred embodiment, the composition according to theinvention is a polyvalent Norovirus vaccine, which comprises

a) at least one, or a plurality, or at least more than one RNA speciescomprising at least one coding region comprising or consisting of atleast one nucleic acid sequence according to any one of SEQ ID NOs:4411-39690, 39713-39746 as described in PCT/EP2017/060673, or a fragmentor variant of any one of these nucleic acid sequences; andb) at least one, or a plurality, or at least more than one adapted RNAspecies comprising at least one coding region encoding at least onepeptide or protein derived from an Norovirus, or a fragment or a variantof said peptide or protein, the at least one coding region preferablycomprising or consisting of at least one adapted nucleic acid sequenceencoding an amino acid sequence according to any one of SEQ ID NOs:1-4410 as described in PCT/EP2017/060673, or a fragment or variant ofany one of these nucleic acid sequences.

According to a particularly preferred embodiment, the compositionaccording to the invention comprises at least two RNA species, whereinthe sequence of at least one RNA species is adapted by altering thenumber of A and/or U nucleotides in the sequence of the at least one RNAspecies with respect to the number of A and/or U nucleotides in theoriginal RNA sequence, wherein at least one RNA species comprises atleast one coding region encoding at least one peptide or protein derivedfrom an influenza virus, or a fragment or a variant of said peptide orprotein. Preferably, the at least one adapted RNA in the inventivecomposition comprises at least one coding region encoding a peptide orprotein derived from an influenza virus, or a fragment or a variant ofsaid peptide or protein. More preferably, the at least one coding regionencodes a peptide or protein derived from an influenza virus asdescribed herein, or a fragment or variant thereof. In some embodiments,the composition of the present invention is an influenza vaccine, morepreferably a polyvalent influenza vaccine.

In a preferred embodiment, the composition comprises at least two RNAspecies as described herein, wherein at least one RNA species comprisesat least one coding region encoding at least one antigen selected fromhemagglutinin (HA) and/or neuraminidase (NA) of an influenza virus, or afragment or variant thereof. More preferably, the at least one codingregion of the RNA encodes at least one antigenic peptide or proteinderived from hemagglutinin (HA) and/or neuraminidase (NA) of aninfluenza virus, or a fragment or variant thereof. In this context, thehemagglutinin (HA) and the neuraminidase (NA) may be chosen from thesame influenza virus or from different influenza viruses (or differentinfluenza virus strains, respectively).

Suitable influenza antigens and antigenic peptides or proteins derivedfrom influenza virus are described in international patent applicationsPCT/EP2016/075862 and PCT/EP2017/060663, which are herein incorporatedby reference in their entirety.

Preferably, the at least one RNA (species), preferably the original RNA(species) that is to be adapted or the adapted RNA (species), in theinventive composition comprises at least one coding region, whichencodes a peptide or protein comprising or consisting of an amino acidsequence according to any one of SEQ ID NOs: 1-30504, 213713, 213738,213739, 213787, 213792, 213797, 213802, 213996-214023, 214100-214127,214212-214239, 214316-214343, 214420-214447, 214524-214551,214628-214655, 214732-214759, 214836-214863, 214940-214967, 215044,215049-215076, 215161, 215166-215193, 215278, 215283-215310, 215395,215400-215427, 215512, 215517-215544 as described in PCT/EP2017/060663,or a fragment or variant of any one of these amino acid sequences.

According to some embodiments, the at least one RNA species, preferablythe original RNA (species) that is to be adapted, in the inventivecomposition comprises at least one coding region comprising orconsisting of a nucleic acid sequence according to any one of SEQ IDNOs: 30505-213528, 213529-213557, 213740-213746, 213788, 213789, 213793,213794, 213798, 213799, 213803, 213804, 214024-214051, 214128-214155,214240-214267, 214344-214371, 214448-214475, 214552-214579,214656-214683, 214760-214787, 214864-214891, 214968-214995, 215045,215046, 215077-215104, 215162, 215163, 215194-215221, 215279, 215280,215311-215338, 215396, 215397, 215428-215455, 215513, 215514,215545-215572, 215629, 215632, 215638-215835, 215892, 215836-215889 asdescribed in PCT/EP2017/060663, or a fragment or variant of any one ofthese nucleic acid sequences.

According to a particularly preferred embodiment, the at least one RNAspecies, preferably an adapted RNA species, in the inventive compositioncomprises at least one coding region comprising or consisting of anucleic acid sequence according to any one of SEQ ID NO: 26 to 14079,14080 to 16264, 16265 to 28640, 28641 to 30568 as disclosed herein, or afragment or variant of any one of these nucleic acid sequences.

In a further preferred embodiment, the composition according to theinvention is a polyvalent influenza vaccine, which comprises at leasttwo RNA species, wherein the sequence of at least one RNA species isadapted by altering the number of A and/or U nucleotides in the sequenceof the at least one RNA species with respect to the number of A and/or Unucleotides in the original RNA sequence, wherein at least one RNAspecies comprises at least one coding region encoding at least onepeptide or protein derived from an influenza virus, or a fragment or avariant of said peptide or protein, and wherein at least one RNA speciescomprises at least one coding region encoding a different peptide orprotein derived from an influenza virus, or a fragment or a variant ofsaid peptide or protein. More preferably, the composition comprises atleast one RNA species encoding a peptide or protein derived frominfluenza HA, or a fragment or variant thereof, and at least one otherRNA species encoding a peptide or protein derived from influenza NA, ora fragment or variant thereof, wherein at least one of said RNA speciesis an adapted RNA as described herein.

In a particularly preferred embodiment, the composition according to theinvention is a polyvalent influenza vaccine, which comprises

a) at least one, or a plurality, or at least more than one RNA speciescomprising at least one coding region comprising or consisting of atleast one nucleic acid sequence according to any one of SEQ ID NOs:30505-213528, 213529-213557, 213740-213746, 213788, 213789, 213793,213794, 213798, 213799, 213803, 213804, 214024-214051, 214128-214155,214240-214267, 214344-214371, 214448-214475, 214552-214579,214656-214683, 214760-214787, 214864-214891, 214968-214995, 215045,215046, 215077-215104, 215162, 215163, 215194-215221, 215279, 215280,215311-215338, 215396, 215397, 215428-215455, 215513, 215514,215545-215572, 215629, 215632, 215638-215835, 215892, 215836-215889 asdescribed in PCT/EP2017/060663, or a fragment or variant of any one ofthese nucleic acid sequences;andb) at least one, or a plurality, or at least more than one adapted RNAspecies comprising at least one coding region encoding at least onepeptide or protein derived from an influenza virus, or a fragment or avariant of said peptide or protein, the at least one coding regionpreferably comprising or consisting of at least one nucleic acidsequence according to any one of SEQ ID NO: 26 to 14079, 14080 to 16264,16265 to 28640, 28641 to 30568 as disclosed herein, or a fragment orvariant of any one of these nucleic acid sequences.

Method for Producing an RNA Composition

A further aspect of the present invention concerns a method forproducing an RNA composition as described herein. The method ispreferably for producing an RNA composition, preferably a (polyvalent)vaccine, comprising at least two RNA species, wherein the sequence of atleast one RNA species has been adapted by altering the number of Aand/or U nucleotides in the sequence of the at least one adapted RNAspecies, preferably as described herein, more preferably as describedherein with respect to the method for modifying the retention time of anRNA on a chromatographic column, to the method for purifying at leastone RNA species from a mixture of at least two RNA species, to themethod for co-purifying at least two RNA species from a mixturecomprising at least two RNA species, to the method for harmonizing thenumbers of A and/or U nucleotides in the sequences of at least two RNAspecies, to the method fro providing an adapted RNA or to thecomposition or vaccine as described herein.

In a preferred embodiment, the method provides a system, which allowsfor fast and efficient production of a composition comprising at leasttwo RNA species, which are selected from a pool of a RNA species.Starting out from a pool of RNA species/sequences, the at least two RNAspecies of the composition can quickly be exchanged by the methods laidout herein. The concept is also illustrated by FIG. 3 and Example 7. Forexample, in the case of a polyvalent influenza vaccine, from an AUadapted RNA sequence pool, RNA species encoding antigens, e.g. HA and/orNA antigens, can easily be exchanged (“+”: addition of new RNA speciesto the RNA mixture; “−”: removal of RNA species from the mixture) fore.g. seasonal influenza vaccine production (see FIG. 3; A: season A; B:season B; C: season C). In that manner, the method allows for quickseasonal adaptation of the influenza vaccine. The outlined concept isalso suitable for other fast-adapting pathogens, e.g. Norovirus, and maybe used to quickly adapt RNA based Norovirus vaccines.

The method may thus preferably involve the steps of adapting thesequence of at least one RNA sequence, which is comprised in thecomposition. Methods for producing RNA are known in the art anddescribed herein.

Kits

According to another aspect, the present invention also provides kits,particularly kits of parts, comprising at least one (adapted) RNA(species) according to the invention, the composition comprising atleast one (adapted) RNA (species) according to the invention, optionallya liquid vehicle for solubilising and optionally technical instructionswith information on the administration and dosage of the (adapted) RNA(species) as described herein. Preferably, the (adapted) RNA (species)as described herein or the composition comprising at least one (adapted)RNA (species) according to the invention is provided in a separate partof the kit, wherein the (adapted) RNA (species) as described herein orthe composition comprising at least one (adapted) RNA (species)according to the invention are preferably lyophilised. More preferably,the kit further contains as a part a vehicle for solubilising the RNA asdescribed herein, the composition comprising at least one RNA (species)according to the invention, the vehicle preferably being Ringer-lactatesolution. Any of the above kits may be used in a treatment orprophylaxis as defined herein. More preferably, any of the above kitsmay be used as a vaccine, preferably a vaccine against infection with aninfectious disease, such as an influenza virus infection or Norovirusinfection.

(Medical) Use and Application:

The present invention furthermore provides several applications and usesof the (adapted) RNA (species) according to the invention, thecomposition/vaccine comprising at least one (adapted) RNA (species)according to the invention or of kits comprising same. In particular,the (pharmaceutical) composition or the vaccine may be used for humanand also for veterinary medical purposes, preferably for human medicalpurposes, as a pharmaceutical composition in general or as a vaccine.

Consequently, in a further aspect, the present invention is directed tothe first medical use of the (adapted) RNA (species) according to theinvention, the composition/vaccine comprising at least one (adapted) RNA(species) according to the invention or the kit or kit of parts asdefined herein as a medicament. In particular, the invention providesthe use of at least one (adapted) RNA (species) as defined herein, or afragment or variant thereof as described herein for the preparation of amedicament.

According to another aspect, the present invention is directed to thesecond medical use of the (adapted) RNA (species) according to theinvention, the composition comprising at least one (adapted) RNA(species) according to the invention or the kit or kit of parts asdescribed herein for the treatment of an infection with an influenzavirus. In particular, the present invention provides the at least one(adapted) RNA (species) as described herein to be used for thepreparation of a medicament, wherein the (adapted) RNA (species) asdescribed herein is preferably formulated together with apharmaceutically acceptable vehicle and an optionally additionaladjuvant and an optionally additional further component as definedherein.

Further, the present invention is directed to the second medical use ofthe (adapted) RNA (species) according to the invention, the compositioncomprising at least one (adapted) RNA (species) according to theinvention or the kit or kit of parts as described herein for thetreatment of an infection with a Norovirus. In particular, the presentinvention provides the at least one (adapted) RNA (species) as describedherein to be used for the preparation of a medicament, wherein the(adapted) RNA (species) as described herein is preferably formulatedtogether with a pharmaceutically acceptable vehicle and an optionallyadditional adjuvant and an optionally additional further component asdefined herein.

The composition or the vaccine comprising at least one (adapted) RNA(species) according to the invention can be administered, for example,systemically or locally. Routes for systemic administration in generalinclude, for example, transdermal, oral, parenteral routes, includingsubcutaneous, intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,the vaccine may be administered by an intradermal, subcutaneous, orintramuscular route.

In a further aspect the invention provides a method of treating orpreventing a disorder, wherein the disorder is preferably an infectionwith influenza virus or Norovirus, or a disorder related to an infectionwith influenza virus or Norovirus, wherein the method comprisesadministering to a subject in need thereof the (adapted) RNA (species)according to the invention, the inventive composition comprising atleast one (adapted) RNA (species) according to the invention, or theinventive kit or kit of parts.

In particular, such a method may preferably comprise the steps of:

a) providing the (adapted) RNA (species) according to the invention, thevaccine/composition comprising at least one (adapted) RNA (species)according to the invention, or the inventive kit or kit of partsdescribed herein; andb) applying or administering the (adapted) RNA (species) according tothe invention, the vaccine/composition comprising at least one (adapted)RNA (species) according to the invention, or the kit or kit of partsdescribed herein to a tissue or an organism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: illustrates the technical problem associated with HPLCco-purification and/or co-analysis of RNA mixtures. Schematic drawingsof HPLC histograms (RNA mixture comprising different RNA moleculespecies) are shown.

FIGS. 1A and 1B: RNA molecule species of an RNA mixture differ in theirretention times. Impurities such as abortive sequences cannot beseparated from each other. In addition, as the histograms partiallyoverlap, quality attributes, such as integrity of the individualspecies, cannot be determined. FIG. 1A shows the separate histograms foreach RNA species in the mixture.

FIGS. 1C and 1D: In rare cases, three RNA species of an RNA mixture mayhave similar retention times, and impurities such as abortive sequencescould be separated from each other. In addition, as the peaks entirelyoverlap, quality attributes, such as integrity of the whole RNA mixture,could be determined. FIG. 1C shows the separate histograms for each RNAspecies in the mixture. FIG. 1E: RNA species have different retentiontimes. As the histograms are entirely separated from each other, qualityattributes, such as integrity of individual RNA species of the RNAmixture, can be determined.

FIG. 2: illustrates the basic principle of adapting a given nucleic acidsequence encoding a short protein (amino acid sequence: AWHPVAC) toeither increase the AU count or decrease the AU count. FIG. 2A: Asinitial step of the adaptation method/algorithm, all codons of thenucleic acid sequence are categorized and potential exchanges withalternative codons are allocated for each individual codon (codonchanges that allow increase or decrease of AU count are listed in Table1 and Table 2). In FIG. 2A, codons labeled with an asterisk (*) mayallow for an increase in AU count if the respective codon is changedaccordingly; codons labeled with a hash (#) may allow for a decrease inAU count if the respective codon is changed accordingly; codons labeledwith a cross (“x”) do not lead to a change in AU count. FIG. 2B showsthe adaptation of the input nucleic acid sequence to an AU countincreased target sequence. All four codons, which can potentially bereplaced by alternative codons, are changed to the codon with a largerAU count (codons highlighted). FIG. 2C shows the adaptation of the inputnucleic acid sequence to an AU decreased target sequence. All threecodons, which can potentially be replaced by alternative codons, arechanged to the codon with a lower AU count (codons highlighted).

FIG. 3: illustrates the modular design principle of the inventivemultivalent/polyvalent mRNA vaccine platform (e.g. influenza), where RNAspecies can be exchanged rapidly without changing the manufacturingconditions (HPLC purification and/or HPLC analysis). From an AU adaptedRNA sequence pool, RNA species encoding antigens, e.g. HA and/or NAantigens, can easily be exchanged (“+”: addition of new RNA species tothe RNA mixture; “−”: removal of RNA species from the mixture) for e.g.seasonal influenza vaccine production (A: season A; B: season B; C:season C). The general concept can also be used for e.g. Norovirusantigens.

FIG. 4: shows that the number of adenine nucleotides in an RNA sequencecorrelates with HPLC retention times. RNA species (1-4) encoding fireflyluciferase with varying polyA sizes were generated and individuallyanalyzed via HPLC. HPLC chromatograms were superimposed. 1=A25; 2=A35;3=A50; 4=A64. A detailed description of the experiment is provided inExample 1.

FIG. 5: shows that adaptation of the number of adenine nucleotides inRNA sequences enables harmonization of HPLC retention time. FIG. 5A:Adenine adapted RNA sequences encoding HA-B Brisbane are shown(superimposed). FIG. 5B: Adenine adapted RNA sequences encoding HA-BPhuket are shown (superimposed). (1) Non-adapted sequence; (2) 9adenines introduced in cds; (3) 9 adenine stretch added in the UTR or(4) 9 adenines added in polyA. A detailed description of the experimentis provided in Example 2.

FIG. 6: shows that adaptation of the adenine count enables harmonizationof HPLC retention time of RNA sequences encoding HA-A and RNA sequencesencoding HA-B. RNA sequences encoding HA-B were adapted to match the Acount of HA-A RNA sequences by increasing the A count accordingly. Theasterisk indicates that the HPLC peaks completely overlap (decliningslope of the peak determines retention time). Of note: For the purposeof FIG. 6, a distinction of individual chromatograms is not required. Adetailed description of the experiment is provided in Example 2.

FIG. 7: shows that HPLC is a particularly suitable method forco-analysis of an RNA mixture. RNA Mixtures of intact and degraded RNAat different ratios were analyzed via HPLC. FIG. 7A: Overlay of HPLCchromatograms of different RNA mixtures showing the amount of intactRNA. FIG. 7B: Overlay of HPLC chromatograms of different RNA mixturesshowing the amount of degraded RNA. Of note: For the purpose of FIG. 7,a distinction of individual chromatograms is not required. A detaileddescription of the experiment is provided in Example 3.

FIG. 8: shows that an adenine adapted RNA mixture (encoding threedifferent NA antigens) generates one discrete HPLC peak, suitable forco-analysis and co-purification. FIG. 8A: HPLC histograms for individualnon-adapted RNA sequences (1-3) and the resulting non-adapted RNAmixture (4) are shown. FIG. 8B: HPLC histogram for individual sequenceadapted RNA sequences (1-3) and the resulting harmonized RNA mixture (4)are shown. A detailed description of the experiment is provided inExample 4.

FIG. 9: shows a further illustration of the technical problem. RNAspecies encoding HA and NA of Influenza virus A and B have partiallyoverlapping HPLC chromatograms due to different AU counts, illustratingthe problem in the art that co-purification and/or co-analysis of suchan RNA mixture (comprising all seven RNA species) would be technicallyimpossible. 1=RNA species encoding neuraminidase of Influenza virus B(NA-B, Brisbane); 2=two RNA species encoding neuraminidase of Influenzavirus A (NA-A, Hongkong, Calif.); 3=two RNA species encodinghemagglutinin of Influenza virus B (HA-B, Brisbane, Phuket); two RNAmolecule species encoding hemagglutinin of Influenza virus A (HA-A,Hongkong, Calif.). A detailed description of the experiment is providedin Example 5.

FIG. 10: shows that adenine and/or uracil count correlates with HPLCretention times.

FIG. 10A: Total number/count of A and/or U and the content (%) of AU ofdifferent HA and NA RNA species plotted against the RNA retention timeon HPLC is shown. The total number/count of A and/or U correlates withHPLC retention time, whereas the content of AU does not correlate withthe HPLC retention time.

FIG. 10B: Total number/count of G and/or C and the content (%) of GC ofdifferent HA and NA RNA species plotted against the RNA retention timeon HPLC is shown. Both guanine/cytosine (G/C) count and content (%) donot correlate with HPLC retention times. A detailed description of theexperiment is provided in Example 5.

FIG. 11: illustrates the sequence adaptation strategy for an RNA mixturecomprising three different RNA molecule species. Two product peaks haveto be shifted by 17 AU or 40 AU (shifted peaks: dashed lines) to obtaina sequence adapted RNA mixture, where each of the three components canbe co-analyzed on HPLC (AU count difference for the adapted sequences:70 AU). A detailed description of the experiment is provided in Example6.

FIG. 12: overlay of HPLC chromatograms of constructs with different AUcount on a monolithic ethylvinylbenzene-divinylbenzene copolymer. Flowrates and gradient profiles indicated. First peak corresponds to aconstruct with AU count 824, second peak corresponds to a construct withAU count 894, third peak corresponds to a construct with AU count 1137,fourth peak corresponds to a construct with AU count 1139, last peakcorresponds to a construct with AU count 1424. A detailed description ofthe experiment is provided in Example 8.

FIG. 13: overlay of HPLC chromatograms of constructs with different AUcount on a particulate poly(styrene)-divinylbenzene (PVD) column. Flowrates and gradient profiles indicated. First peak corresponds to aconstruct with AU count 824, second peak corresponds to a construct withAU count 894, third peak corresponds to a construct with AU count 1137,fourth peak corresponds to a construct with AU count 1139, last peakcorresponds to a construct with AU count 1424. A detailed description ofthe experiment is provided in Example 8.

FIG. 14: overlay of HPLC chromatograms of constructs with different AUcount on a Silica-based C4 column. Flow rates and gradient profilesindicated. First peak corresponds to a construct with AU count 824,second peak corresponds to a construct with AU count 894, third peakcorresponds to a construct with AU count 1137, fourth peak correspondsto a construct with AU count 1139, last peak corresponds to a constructwith AU count 1424. A detailed description of the experiment is providedin Example 8.

FIG. 15: overlay of HPLC chromatograms of constructs with different AUcount on a PLPR-S column. Flow rates and gradient profiles indicated.First peak corresponds to a construct with AU count 824, second peakcorresponds to a construct with AU count 894, third peak corresponds toa construct with AU count 1137, fourth peak corresponds to a constructwith AU count 1139, last peak corresponds to a construct with AU count1424. A detailed description of the experiment is provided in Example 8.

FIG. 16: plot of separation factor alpha against AU count difference forrepresentative HPLC runs of Example 8. Monolithic: Values and respectivetrend line for monolithic ethylvinylbenzene-divinylbenzene copolymerwith flowrate 0.5, cf. FIG. 12; PVD: Values and respective trend linefor particulate poly(styrene)-divinylbenzene (PVD) column with flowrate0.3, cf. FIG. 13; C4-F1: Values and respective trend line forsilica-based C4 column with flowrate 1.0, cf. FIG. 14; PLPR-S: Valuesand respective trend line for PLPR-S column with flowrate 1.0, cf. FIG.15. A detailed description of the experiment is provided in Example 8.

EXAMPLES

The Examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These Examples shallnot be construed to limit the present invention thereto.

TABLE 3 Materials used U3000 UH PLC-System Thermo Scientific HPLC columnpoly(styrene- Thermo Scientific divinylbenzen) matrix) WFI FreseniusKabi, Ampuwa Acetonitril (MS-grade) Fisher Scientific 0.1M TEAA in WFI(Eluent A) 25% ACN in 0.1M TEAA (Eluent B)

Example 1: Examination of the Correlation Between Homopolymer Stretchesof Nucleotides on HPLC Retention Times

The inventors surprisingly found that not the size of an RNA, but thetotal number of adenine nucleotides (A nucleotides) and/or uracilnucleotides (U nucleotides) of an RNA is influencing HPLC retentiontimes. Further details are provided in the following.

1.1. Preparation of DNAs Encoding Firefly Luciferase Including VaryingStretches of Adenines:

The DNA sequence encoding firefly luciferase protein was introduced intoa modified pUC19 derived vector backbone to comprise a 5′-UTR derivedfrom the 32L4 ribosomal protein (32L4 TOP 5′-UTR) and a 3′-UTR derivedfrom albumin, a histone-stem-loop structure, and stretches of varyingnumbers of adenine nucleotides (also referred to in the following as‘polyA stretch’ or ‘A homopolymer’) at the 3′-terminal end and. Thecomplete RNA sequences are provided in the sequence listing (see Table 4below).

TABLE 4 Constructs used in the experiment Encoded Length of proteinpolyA stretch SEQ ID NO: luciferase A25 1 A35 2 A50 3 A64 4

DNA plasmids were linearized using EcoRI and transcribed in vitro usingDNA dependent T7 RNA polymerase in the presence of a nucleotide mixtureand cap analog under suitable buffer conditions. The obtained individualRNA products were purified using PureMessenger® as described in WO2008/077592 A1 and subsequently analyzed using HPLC.

1.2 Determination of HPLC Retention Times:

Individual RNA samples were diluted to 0.1 g/L using water for injection(WFI). 10p1 of the diluted RNA sample were injected into the HPLC column(monolithic poly(styrene-divinylbenzen) matrix). The RP HPLC analysiswas performed using the following conditions:

Gradient 1: Buffer A (0.1 M TEAA (pH 7.0)); Buffer B (0.1 M TEAA (pH7.0) containing 25% acetonitrile). Starting at 30% buffer B the gradientextended to 32% buffer B in 2 minutes, followed by an extension to 55%buffer B over 15 minutes at a flow rate of 1 ml/min (adapted from WO2008/077592). Chromatograms were recorded at a wavelength of 260 nm.

In order to examine an eventual correlation between the presence and theextent of nucleotide homopolymer stretches on the one hand and the HPLCretention time on the other hand, chromatograms of each HPLC analysisrun were superimposed. FIG. 4 shows the superposition of HPLC runs ofRNAs with varying stretches of adenine nucleotides.

Results

As shown in FIG. 4, the analysis of the HPLC retention time of differentRNA molecule species differing only in the length of A nucleotidehomopolymer stretches (i.e. in the total number of A nucleotides) show aclear correlation of the total number of A nucleotides in the RNAsequences and HPLC retention time. Longer A homopolymers led to anincrease in retention time, suggesting that the observed effect on HPLCretention time is caused by the increased number of A nucleotides (+25adenine nucleotides; +35 adenine nucleotides; +50 adenine nucleotides;+64 adenine nucleotides).

Notably, changes in the total number of cytosine nucleotides (Cnucleotides) did not have an influence on HPLC retention time (notshown). As only the number of A nucleotides and not the number of Cnucleotides influences HPLC retention times, an effect merely caused byelongation of the RNA molecule species can be ruled out.

Example 2: Harmonization of HPLC Retention Times of Different HA RNASequences for Co-Purification and/or Co-Analysis by Adaptation of theAdenine Count

The inventors surprisingly found that the adaptation the total number ofA nucleotides in two or more different RNA species (e.g. RNA moleculescomprising different sequences encoding influenza HA-B) is suitable toharmonize the HPLC retention times, so that co-purification and/orco-analysis becomes feasible. Further details are provided in thefollowing.

2.1. Adaptation of the Total Number of a Nucleotides:

As the previous examples show a correlation between the number of Anucleotides in an RNA sequence and the respective HPLC retention time,RNA sequences encoding HA antigens (four different RNA sequencesencoding influenza HA) were adapted so that they comprised (essentially)the same number of A nucleotides. The sequence adaptation was performedin such a way that the encoded amino acid sequence was unchanged, eitherby exploiting the degeneracy of the genetic code (compare with Table 1and Table 2) or by introducing an adenine stretch into the polyA tail orthe UTR of the RNA molecule species.

The goal was to adapt the sequences in a way to facilitateco-purification and/or co-analysis of an RNA mixture comprisingdifferent HA RNA molecule species by obtaining a complete overlay of thefour chromatograms (harmonization) in HPLC, which is a prerequisite fora cost-effective and fast production of an influenza vaccine based on anmRNA mixture (e.g., for the development of a multivalent/polyvalentinfluenza RNA vaccine platform, cf. FIG. 3).

In order to harmonize the retention times of all RNA molecule speciesencoding different HA antigens (HA-A and HA-B), GC-optimized DNAsequences encoding different HA proteins of Influenza B were adapted byincreasing the number of A nucleotides by adapting the coding sequence(via codon exchange), by elongating the poly A sequence, or byintroducing additional A nucleotides into the UTR region (see Table 5below). The adaptation was performed by increasing the total number of Anucleotides in the HA-B sequences by 9 in order to shift the totalnumber of A nucleotides in the HA-B sequences closer to the number of Anucleotides in the HA-A sequences. DNA constructs and RNA prepared asexplained in Example 1.

TABLE 5 HA-constructs used in the experiment Encoded A count AU countSEQ ID Antigen Mode of adaptation of RNA* of RNA** NO: HA-B Not adapted467 723 5 Brisbane 9 A nucleotides introduced 476 732 6 into cds bycodon exchange 9 A stretch introduced into poly A tail 476 732 7 9 Astretch introduced into the UTR 476 734 8 HA-B Not adapted 458 717 9Phuket 9 A nucleotides introduced into 467 726 10 cds by codon exchange9 A stretch in poly A tail 467 726 11 9 A stretch introduced into theUTR 467 728 12 HA-A Not adapted 476 737 13 California HA-A Not adapted481 729 14 Hongkong *A-count of RNA: total number of A nucleotides inthe respective RNA **AU-count of RNA: total number of A and Unucleotides in the respective RNA

2.2. Effect of the Total Number of a Nucleotides on HPLC Retention Time:

HPLC sample preparation and HPLC analysis were performed as describedExample 1. In order to examine the effect of the number of A nucleotideson HPLC retention time, the chromatograms of each RNA species weresuperimposed and analyzed.

FIG. 5A shows four superimposed chromatograms for RNA molecule speciesencoding HA-B/Brisbane (one non-adapted sequence (1) and three adaptedsequences (2, 3 and 4, respectively)). FIG. 5B shows four superimposedchromatograms for the RNA molecule species encoding HA-B/Phuket (onenon-adapted sequence (1) and three adapted sequences (2, 3 and 4,respectively)). FIG. 6 shows superimposed chromatograms for adapted RNAmolecule species (9 Adenines introduced into cds by codon exchange)encoding HA-B/Brisbane, adapted RNA molecule species encodingHA-B/Phuket and two non-adapted RNA molecule species encoding HA-A (HA-ACalifornia; HA-A Hongkong).

Results:

The results show that the adaptation of the number of A nucleotides inthe RNA sequences (see FIG. 5A and FIG. 5B) by addition of 9 adeninesled to a shift in HPLC retention time. As indicated in FIG. 5A and FIG.5B, the effect of an A-stretch, either introduced into the UTR orintroduced into the polyA tail had a slightly stronger effect on theretention time.

FIG. 6 shows that HA-B sequences were successfully adapted, and thatHA-A peaks and HA-B peaks are harmonized. This adaptation of thesequences, which results in completely overlapping HPLC peaks, allowsfor co-analysis of the individual RNA species in the mixture and forsimultaneous determination of the integrity of the RNAs in the mixture.Moreover, harmonization of HPLC retention times allows for asimultaneous RNA purification (co-purification).

Of note, as analyzed and explained in further detail in Example 5, thesurprisingly precise overlap of the HA-A sequences and adapted HA-Bsequences as observed in FIG. 6 can also be explained by the closelymatching number of A nucleotides and U nucleotides (AU count) of therespective RNA sequences (HA-B Brisbane: AU count 732; HA-B Phuket: AUcount 726; HA-A California: AU count 737; HA-A Hongkong: AU count: 729;see Table 4).

Example 3: Evaluation of Suitability of HPLC for Co-Analysis of RNAMixture

The inventors showed that HPLC is a particularly suitable method forco-analysis of an RNA mixture. Further details are provided in thefollowing.

3.1. Preparation of Test RNA:

RNA for testing the HPLC system was generated according to Example 1.

3.2. Directed Degradation of RNA and Preparation of RNA Mixtures ofDifferent Integrities:

RNA samples were degraded at 90° C. for 140 minutes. Subsequently,intact RNA and degraded RNA were mixed in different ratios of intactRNA: degraded RNA (90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70,20:80, and 10:90) and respective RNA mixtures of varying integritieswere applied to analytic HPLC. Analytic HPLC was performed as describedin Example 1. For analysis, HPLC runs of the different RNA mixtures weresuperimposed. The results are shown in FIG. 7.

Results:

As FIG. 7 shows, RNA integrity of an RNA mixture can be determined in ascenario where the RNA mixture has the same retention time (harmonizedRNA peak; peak of the individual RNA components, in that case integerRNA and degraded RNA, are completely overlapping). Accordingly, theanalytic system is suitable for the analysis of a (polyvalent) RNAmixture according to the present invention.

Example 4: Harmonization of HPLC Retention Times of NA RNA Sequences forCo-Purification and/or Co-Analysis by Adaptation of the Number of aNucleotides

The inventors surprisingly found that an RNA mixture (encoding threedifferent NA antigens) comprising RNA species with an adapted number ofA nucleotides generates one harmonized HPLC peak, suitable forco-analysis and co-purification. Further details are provided in thefollowing.

4.1. Adaptation of the Number of a Nucleotides in DNA Encoding NAProteins of Several Influenza Strains:

The goal was to adapt NA RNA sequences in a way to facilitateco-purification and/or co-analysis of an RNA mixture of different NA RNAmolecule species by obtaining a complete overlay of the threechromatograms, which is a prerequisite for a cost-effective and fastproduction of an RNA-mixture based influenza vaccine (e.g. for thedevelopment of a multivalent influenza RNA vaccine, cf. FIG. 3)

In order to harmonize the retention time of all RNA molecule speciesencoding different NA antigens, GC-optimized DNA sequences encodingdifferent NA proteins of Influenza were adapted by decreasing the numberof A nucleotides by altering the coding sequence (codon exchange; seeTable 6 below). The adaptation was performed in order to decrease thenumber of A nucleotides in RNA encoding NA H3N2 and mRNA encoding NAH1N1 to essentially match the number of A nucleotides in RNA encoding NAInfluenza B.

DNA constructs and RNA were prepared as explained in Example 1.

TABLE 6 NA-constructs used in the experiment Encoded Antigen Mode ofadaptation SEQ ID NO: NA Influenza B Not adapted 15 (Brisbane) NA H3N2Not adapted 16 (Hongkong) 17 A removed from cds 17 by codon exchange NAH1N1 Not adapted 18 (California) 16 A removed from cds 19 by codonexchange

4.2. Effect of the Number of a Nucleotides on HPLC Retention Time:

HPLC sample preparation and HPLC analysis were performed as described inExample 1.

In order to examine the effect of the number of A nucleotides on HPLCretention time, the chromatograms of non-adapted RNA species weresuperimposed and analyzed. In addition, non-adapted RNA molecule specieswere mixed (100 ng each), applied as a mixture, and analyzed by HPLC(see FIG. 8A). In addition, adapted NA H3N2 (Hongkong) RNA, adapted NAH3N2 (California) RNA, and NA Influenza B (Brisbane) RNA were mixed (100ng each) and applied as a mixture, and analyzed by HPLC (see FIG. 8B).FIG. 8A shows superimposed chromatograms for non-adapted RNA moleculespecies encoding NA (1, 2, 3) next to the chromatogram of thecorresponding RNA mixture (4). FIG. 8B shows the chromatogram of theharmonized NA RNA mixture (4).

Results

The results show that the adaptation of the number of A nucleotides inthe individual RNA sequences of an RNA mixture leads to adaptation ofthe retention time of the RNA mixture and a discrete HPLC peak (see FIG.8B), which allows for co-analysis and co-purification (even though theindividual HPLC peaks show a slight variation). In contrast, anon-adapted RNA mixture generates a broad, non-discrete HPLC double-peak(see FIG. 8B) that is not suitable for co-analysis and co-purification.

Of note, the adaptation (reduction) of the number of A nucleotides inSEQ ID NOs: 17 and 19 was performed by changing serine codon AGC tocodon UCC, which led to a decrease in A count and to an increase in Ucount (AU count was therefore stable; ratio of A:U was decreased),suggesting that the observed slight variation in the HPLC chromatogramsof the individually analyzed adapted sequences was caused by a shift inthe A:U ratio. Accordingly, adaptation of the A:U ratio can also be usedfor sequence adaptations according to the invention.

Example 5: Examination of the Influence of Nucleotides on HPLC RetentionTime

As shown in the previous examples, the adaptation of the number of Anucleotides in RNA sequences allows for harmonization of HPLCchromatograms, which is a requirement for co-analysis and/orco-purification. The inventors further found that the number of A and/orU nucleotides correlates with HPLC retention time. That finding provideseven more options for adapting an RNA sequence and to harmonize HPLCchromatograms of RNA mixtures. Further details are provided in thefollowing.

5.1. Preparation of DNA Encoding HA Proteins of Several InfluenzaStrains:

DNA sequences encoding different haemagglutinin (HA) and neuraminidase(NA) proteins, two glycoproteins found on the surface of influenzaviruses (Influenza A and Influenza B), were generated, and RNA wasproduced as described in Example 1.

TABLE 7 HA-constructs used in the experiment: Encoded antigen SEQ ID NO:HA-A California 13 HA-A Hongkong 14 HA-B Brisbane 5 HA-B Phuket 9 NAH1N1 (California) 18 NA H3N2 (Hongkong) 16 NA Influenza B (Brisbane) 15

5.2. Correlation Between the Total Number of a Nucleotide and/or theRelative Content of a Nucleotide and the HPLC Retention Time:

HPLC sample preparation and HPLC analysis were performed as describedExample 1.

In a first step, the individually produced RNA constructs (RNA species)encoding HA and NA antigens were separately analyzed on HPLC. Thesuperimposed HPLC chromatograms are shown in FIG. 9. The superimpositionof the chromatograms of the seven different RNA species showed that allchromatograms partially overlap, which makes both, co-purification andco-analysis via HPLC technically impossible (compare illustration ofproblem in the art, FIG. 1).

For a better understanding of the impact of the nucleotide sequence onHPLC retention time, the correlation between the nucleotide count(A,U,G, and C) and nucleotide content for each RNA molecule species andHPLC retention time was examined.

FIG. 10A shows the correlation between number and content (AU %) of Aand U nucleotides of different HA and NA RNA species and theirrespective HPLC retention times. FIG. 10B shows the correlation betweennumber and content (GC %) of guanine (G) and cytosine (C) nucleotides ofdifferent HA and NA RNA species and their respective HPLC retentiontimes.

Results:

FIG. 10A shows a clear correlation of the number of A and/or Unucleotides with the respective retention time. Such a correlation wasnot found for the content of A and U nucleotides (AU %). FIG. 10B showsHPLC retention times are neither influenced by the number of G and/or Cnucleotides, nor by the content of G and/or C nucleotides (GC %).

Notably, the correlation between the number of A nucleotides and theretention time is stronger than the correlation between the number of Unucleotides and the retention time; In line with that, the results ofExample 4 also suggested that the effect of A nucleotides on retentiontime is stronger than the effect of U nucleotides on retention time.

Overall, the number of A and U nucleotides shows the best correlationand will allow for the most precise way for adapting RNA sequences toharmonize RNA mixtures for co-analysis and co-purification.

Example 6: Development of an Automated Nucleotide Adaptation Method(Algorithm)

The inventors developed an automated in silico method (algorithm) to setthe number of any nucleotide in an RNA sequence to a certain definedvalue, without altering the amino acid sequence. In the context of theinvention, the automated in silico method was used for sequenceadaptation (adaptation of the number of A and/or U nucleotides (AUcount)) of RNA sequences to allow harmonization of RNA mixtures for HPLCco-analysis and/or HPLC co-purification. Further details are provided inthe following.

6.1 Sequence Analysis and Definition of Target AU Count:

The objective of the experiment was to generate RNA sequences for anadapted RNA mixture (comprising three different RNA molecule speciesencoding antibodies) suitable for co-analysis using HPLC. The AU counthas to be adapted in all RNA molecule such that their respective HPLCchromatograms are completely separated (difference in the AU count of atleast 70), allowing for co-analysis of their integrity.

Three antibody sequences (SEQ ID NOs: 20-22) were selected and GCoptimized DNA (SEQ ID NOs: 23-25) sequences were generated (essentiallyaccording to Example 1). Nucleotide numbers were determined for therespective GC optimized sequences (product 1, product 2, product 3; seeTable 8 below) to be able to define optimal numbers of A and U (T)nucleotides for HPLC co-analysis.

TABLE 8 Nucleotide numbers for GC optimized constructs: product Length Acount T (U) count AT (AU) count SEQ ID NO: 1 81 19 13 32 23 2 258 59 2685 24 3 429 77 55 132 25

To adapt the RNA molecule species comprised in the RNA mixture for HPLCco-analysis, the target AU counts for product 2 and product 3 were setto the following values, allowing integrity analysis on HPLC whenanalyzed as an RNA mixture:

TABLE 9 Adaptation strategy for co-analysis of the RNA mixture: AT (AU)count Change in Target AT product (non-adapted) AU count (AU) count 1 320 32 2 85 +17 102 3 132 +40 172

As indicated in Table 9, the target AU count for each product RNA wasset in such a manner that the AU counts of the three RNA sequencesdiffer by at least 70 nucleotides (strategy illustrated in FIG. 11).

6.2 AU Sequence Adaptation Method:

In the following, the sequence adaptation method is exemplarilydescribed for product 2 (+17 AU) (SEQ ID NO: 24). As the number of Anucleotides in the sequence was larger than the number of T (U)nucleotides, the adaptation values were set to +8A and +9T(U) in orderto maintain the distribution of A and U nucleotides in the resulting AUadapted sequence.

In the initial phase of the method (algorithm), a matrix for each codoncomprised in the sequence was created, identifying possible changes(herein referred to as “exchange matrix”). An exemplary “exchangematrix” is shown in Formula (I).

$\begin{matrix}{\left. \begin{matrix}A & 1 \\C & 1 \\G & 1 \\T & 1 \\* & 4\end{matrix} \right\}{CGA}} & {{Formula}\mspace{14mu}(I)}\end{matrix}$

Formula (I) shows that for codon “CGA” a change to an alternative codonoffers the option of increasing the number of A nucleotides by 1 (e.g.:CGA->AGA), offers the option of increasing the number of C nucleotidesby 1 (e.g. CGA->CGC), offers the option of increasing the number of Gnucleotides by 1 (e.g. CGA->CGG), and offers the option of increasingthe number of T nucleotides by 1 (e.g. CGA->CGT).

Exchange matrices were generated for each individual codon in thesequence. Using said exchange matrices, the potential maximum number ofthe respective nucleotides (A and T(U) count, respectively) in eachcodon was determined (without changing the amino acid sequence).Accordingly, all 63 codons of the sequence were analyzed, and thepotential alternative codons were assembled in a table structure asshown in Table 10 by way of example.

TABLE 10 Exemplary table of alternative codons allowing for a change inthe number of a nucleotide Codons Alternative codons CGA CGT, CGC, CGG,AGA, AGG GAT GAC GAC GAT ATG no alternative codon . . . . . .

Next, the sequence according to SEQ ID NO: 24 was iteratively dividedinto separate codons and stored in table format, which resulted in alist as exemplarily shown in Table 11 (positions 1, 2, 3, 4 . . . 86 and87 of the sequence are indicated).

TABLE 11 Codon list of SEQ ID NO 24: Codon position Codon 1 ATG 2 AGC 3ATC 4 ATC . . . . . . 86 GAG 87 AGC

Next, the list of codons (see Table 11) was analysed for possible codonchanges by step-wise iteration, wherein in each iteration step thecorresponding codon was analysed using the respective exchange matrix(as outlined above) for potential nucleotide changes. For example, if nochanges were theoretically possible in the respective codon, e.g. as inthe case of “ATG” or “TGG”, the corresponding exchange matrix asexemplarily shown in Formula (II) was used (* of exchange matrix=0).

$\begin{matrix}{\left. \begin{matrix}A & 0 \\C & 0 \\G & 0 \\T & 0 \\* & 0\end{matrix} \right\}{ATG}} & {{Formula}\mspace{14mu}({II})}\end{matrix}$

Formula (II) shows that for codon “ATG” a change to an alternative codonoffers no option of increasing the number of A nucleotides, Cnucleotides, G nucleotides or T nucleotides (as there are no alternativecodons for ATG (Met)).

In cases where changes according to the respective exchange matrix (*>0)were theoretically possible, the codon was further analysed if thesechanges can be implemented under the premise that e.g. only codons thatoffer the option of increasing the number of A and/or T(U) nucleotideswere adapted. Therefore the intersection between the target nucleotides(e.g. A and/or T(U)) and the nucleotides that potentially generate apositive result (that is, A and/or T(U) change; see e.g. Formula (I)) inthe current exchange matrix was constructed. As a result, each codon wascategorized and grouped in three categories:

Category 1 (Category “Favourable”):

Potential codon exchanges allowing an increase in only one targetnucleotide (in the present example A or T(U)). For example, the codon“GAC” (Asp) can be changed to “GAU” (Asp) in order to increase thenumber of A and T(U) nucleotides. No further analysis is required sincethat modification does not have any further impact (besides the onementioned above) on the number of A and T(U) nucleotides.

Category 2 (Category “Possible”):

Potential codon exchanges allowing the increase in both targetnucleotides (in the present example A and T(U)). For example, codon“GCA” can be changed to “GCU”, which would increase the T(U) count butat the same time decrease the A count. Accordingly, further analysiswould be required with respect to codons belonging to this the categoryin order to decide, whether the number of one of the two targetnucleotides (T(U)) in this example should be increased at the expense ofa reduction of the number of the other target nucleotide (A).

Category 3 (Category “Impossible”):

Codons in the RNA sequence, for which no alternative codons exist (*=0).Examples for this category 3 are ATG (Met; start codon) or “UGG” (Trp).

All codons of the original sequence were categorized in that manner.After this step, there were three categories with a total of 87 entries(for 87 codons present in SEQ ID NO: 24). For the next step, category 3was no longer considered, as a codon change will not influence thetarget nucleotide count (A, T(U)).

Next, it was calculated how many potential nucleotide changes have beenidentified for all target nucleotides (A, T(U)). For the SEQ ID NO: 24the possible nucleotide changes are listed (category 1, category 2; seeTable 12).

TABLE 12 Nucleotide changes that can potentially be applied to SEQ ID NO24 Potential Nucleotide changes Category A 22 1 T 15 1 A, T 47 2

Accordingly, 22 favourable changes (see category 1/“favourable” asexplained above) were identified for A and 15 favourable changes wereidentified for T(U). As the adaptation values were set to +8A and +9T(U)the adaptation at codon positions were all taken from category 1. Table13 summarizes the introduced codon exchanges that were equallydistributed across the sequence.

TABLE 13 Codon exchanges introduced into SEQ ID NO 24 Position APosition T(U) A Exchange increase T Exchange increase 30 AAG -> AAA +1 3AGC -> AGT +1 48 AAG -> AAA +1 12 AGC -> AGT +1 78 AAG -> AAA +1 63 GAC-> GAT +1 141 CAG -> CAA +1 90 AGC -> AGT +1 165 GAG -> GAA +1 102 AGC-> AGT +1 213 GAG -> GAA +1 117 AAC -> AAT +1 234 GAG -> GAA +1 138 AGC-> AGT +1 252 GAG -> GAA +1 150 AAC -> AAT +1 180 AGC -> AGT +1

These exchanges resulted in the following adapted sequence according toSEQ ID NO: 25.

In the above described example, potential nucleotide exchanges fromcategory 2 were not implemented. However, in scenarios where e.g. T(U)counts larger than 15 are needed, codons from category 2 are used assoon as all codons from category 1 have been used, in order to obtainadditional adaptation possibilities for A and T(U) counts. If category 2is required in order to achieve the desired nucleotide counts,calculation of the following ratio will identify the exchange nucleotide(nucleotide A or T(U)):

$\frac{c_{i}}{x_{i} - p_{i}}$

wherein i represents the corresponding target, c_(i) is the count ofpossible adaptation positions of i in category 2, x_(i) is the desiredthreshold for i, p_(i) the count for the already changed identifiedadaptation positions. All calculated ratios are ranked and starting fromlowest to highest ratio, the changes from category 2 are applied, untilthe desired threshold has been reached or until all the possibleexchanges from category 2 have been performed. This procedure is carriedout iteratively for all targets, where the desired numbers cannot beachieved by only using exchanges according to category 1.

For example, category 2 contains 47 codons (see Table 12), which couldpotentially be exchanged in order to increase the A or T count.Accordingly, further changes are implemented from category 2 until thedesired threshold has been reached or until all the codons from category2 have been used as well. For SEQ ID NO: 24, a change of the T(U) countto e.g. 20 would result in additional adaptations by using the followingalternative codons from category 2 (additional codon exchanges fromcategory 1 are not shown): 6=ATT, 9=ATT, 21=CTT, 144=CCT, 183=TCT.

In cases where the desired target nucleotide count cannot be achieved(as all alternative codons from category 1 and 2 have been used, whichmeans that no further changes are possible), an adapted sequence isgenerated that is matching the target nucleotide count as close aspossible.

In order to further optimize the above described method (algorithm), thefollowing improvements are implemented:

-   1. The basic equal distribution, which was used in the experiments    described above, is based on the exchange possibilities. Other    distribution models may also be envisaged, such as normal    distribution, first occurrences distribution, last occurrences    distribution or random-based distribution. Alternatively, the mean    of the possible changes or median of the possible changes may be    determined and all exchanges may be arranged around these values.-   2. The exchange matrix contains additional information about the    codon for the target sequence (e.g. codon usage etc.). This creates    a further criteria for the question of whether a codon exchange is    desirable or not, facilitating adaptation to a specific codon usage    or a different nucleotide ratio in the target sequence.-   3. Implementation of a third category by sequences or motifs which    should be avoided by an exchange (e.g. a recognition motif of a    restriction enzyme, promotor sequences or sequences building not    desired secondary structures, etc.).-   4. Automated binning of input sequences, based on their length and    the occurrence of the desired target nucleotides in order to    identify optimal nucleotide counts for A and/or U adaptation.

Example 7: Generation and Use of a Polyvalent Influenza Virus RNAPlatform for Fast-Adjustable Influenza Vaccine Production

A pool of GC optimized coding sequences encoding HA antigens were AUadapted to a count of 612 AU (360 A and 252 T) resulting in an AUadapted HA sequence pool (SEQ ID NO: 26 to 16263). A pool of GCoptimized coding sequences encoding NA antigens were AU adapted to an AUcount of 488 AU (271A and 217U) resulting in an AU adapted NA sequencepool (SEQ ID NOs: 16264-30567). AU adaptations were performed accordingto the invention, essentially as described in Example 6. The adaptationallows co-purification of RNA mixtures comprising adapted HA RNAsequences and co-purification of RNA mixtures comprising adapted NAsequences. Moreover the adaptation allows co-analysis of an RNA mixturecomprising adapted HA and NA RNA species as the RNA sequences encodingHA (AU count 612) and the RNA sequences encoding NA (AU count 488)generate separated peaks (AU count difference: 124), suitable foranalysis of integrity using HPLC.

HA RNA mixtures are produced according to procedures as disclosed in thePCT application WO2017/109134 using GC optimized AT adapted DNAtemplates (generated as described in Example 1). In short, a DNAconstruct mixture (each of which comprising different HA codingsequences and a T7 promotor) is used as a template for simultaneous RNAin vitro transcription to generate a mixture of HA mRNA constructs.Subsequently, the obtained harmonized RNA mixture is used forco-purification using RP-HPLC.

In a parallel reaction, NA RNA mixtures are produced according toprocedures as disclosed in the PCT application WO 2017/109134 using GCoptimized AT adapted DNA templates. In short, a DNA construct mixture(each of which comprising different NA coding sequences and a T7promotor) is used as a template for simultaneous RNA in vitrotranscription to generate a mixture of HA mRNA constructs. Subsequently,the obtained harmonized RNA mixture is used for co-purification usingRP-HPLC.

The purified mRNA mixture encoding HA antigens and the purified mRNAmixture encoding NA antigens are mixed to generate a HA/NA RNA mixture.The integrity of the mixture (that is of the NA peak and the HA peak) isco-analyzed via HPLC as described herein.

Advantageously, the AU adaptation of HA sequences and NA sequences inorder to harmonize chromatographic peaks for HPLC based co-purificationand co-analysis according to the invention facilitates the production ofmRNA-based multivalent influenza vaccines, which may be quickly adaptedto demand, e.g. in seasonal influenza vaccine design or in a pandemicscenario (compare with FIG. 3).

Example 8: Suitability of the Method on Various Reverse Phase HPLCMatrices

The inventors found that modification of the retention time of an RNAvia adaptation of A and/or U count is not restricted to a certainreverse phase column chemistry.

To test whether a modification in retention time via an adaptation of Aand/or U count can also be observed on other reverse phase columns (inExample 1-7, a monolithic poly(styrene-divinylbenzene)matrix has beenused), the following columns were tested:

-   -   monolithic ethylvinylbenzene-divinylbenzene copolymer        (ThermoFisher Scientific) (see FIG. 13)    -   particulate poly(styrene)-divinylbenzene column (ThermoFisher        Scientific) (see FIG. 14)    -   Silica-based C4 column (ThermoFisher Scientific) (see FIG. 15)    -   PLRPS, non-alkylated porous        poly(styrene-divinylbenzene)matrix(see FIG. 16)

RNA encoding yellow fever virus antigens was generated. The constructsencode the same antigen (YFV(17D)-prME), comprise the same UTR elements,and have the same size. The AU count for each constructs was changed viacoding-sequence adaptation. The constructs are listed in Table 14.

TABLE 14 AU adapted YFV constructs SEQ ID NO Antigen RNA size A U G C AUGC 30588 prME 2311 474 350 544 943  824 1487 30589 prME 2311 587 307 704713  894 1417 30590 prME 2311 633 504 627 547 1137 1174 30591 prME 2311788 351 555 617 1139 1172 30592 prME 2311 886 538 516 371 1424  887

To evaluate the effect of AU adaptation on retention time, 500 ug ofeach construct was subjected individually to the respective column. Inaddition, for each column two different flow-rates were tested. Resultsare shown in FIGS. 12-15.

In addition, the separation factor alpha was determined for eachconstruct on each column tested. In chromatography, the separationfactor alpha expresses the ratio of retention times of two compounds.Accordingly, a separation factor of value of larger 1.0 means thatseparation of two compounds occurred. In the present analysis, SEQ IDNO: 30592 (with AU 1424) was taken as a reference for separation factorcalculation. The calculated separation factors are shown in Table 15.The obtained separation factor values were plotted against the AU countdifference of the constructs (SEQ ID NO: 30592, with AU 1424 was takenas a reference). The diagram is shown in FIG. 16.

TABLE 15 AU adapted YFV constructs SEQ RNA AU AU Alpha Alpha Alpha AlphaID NO size count difference monolithic PVD C4 PLPR-S 30592 2311 1424  01,00 1,00 1,00 1,00 30591 2311 1139 285 1,07 1,05 1,05 1,08 30590 23111137 287 1,09 1,07 1,06 1,11 30589 2311  894 530 1,15 1,12 1,10 1,1830588 2311  824 600 1,19 1,15 1,12 1,24

CONCLUSION

As shown in FIG. 13, the modulation of retention time works on amonolithic ethylvinylbenzene-divinylbenzene-copolymer. All fourconstructs elute at different retention times and could be clearlyseparated. In the present case, the separation of construct with AU824from the construct with AU894 was strong enough to allow furtheranalysis of the peaks.

As shown in FIG. 14, the modulation of retention time works on aparticulate poly(styrene)-divinylbenzene column. All four constructselute at different retention times and could be clearly separated. Inthe present case, the separation of construct with AU824 from theconstruct with AU894 was strong enough to allow further analysis of thepeaks.

As shown in FIG. 15, the modulation of retention time works on aSilica-based C4 column column. All four constructs elute at differentretention times and could be clearly separated. In the present case, theseparation of construct with AU824 from the construct with AU1139 wasstrong enough to allow further analysis of the peaks.

FIG. 16 summarizes the inventive concept of the present invention. Anadaption of the AU count of the different RNA constructs (in otherwords: increasing the difference in AU count between the constructs) ledto a modification in retention time, thereby allowing a separation onHPLC. Notably, that effect was observed irrespective of the columnmatrix used.

Of course, a harmonization of AU counts (in other words: decreasing thedifference in AU count between the constructs) would also lead to amodification in retention time, thereby allowing co-purification onHPLC.

1. Method for modifying the retention time of RNA on a chromatographiccolumn, wherein the method comprises a step of adapting the RNA sequenceby altering the number of A and/or U nucleotides in the RNA sequencewith respect to the number of A and/or U nucleotides in the original RNAsequence. 2-28. (canceled)
 29. Method for synthesis of a mixturecomprising at least two harmonized RNA species, the method comprising a)a step comprising harmonizing the numbers of A and/or U nucleotides inthe sequences of at least two RNA species, wherein the sequence of atleast one RNA species is adapted by altering the number of A and/or Unucleotides in the RNA sequence with respect to the number of A and/or Unucleotides in the original RNA sequence; and b) a step of synthesis ofthe at least two harmonized RNA species.
 30. The method according toclaim 29, wherein step b) comprises the separate synthesis of the atleast two harmonized RNA species.
 31. The method according to claim 30,wherein step b) comprises mixing the at least two harmonized RNAspecies.
 32. The method according to claim 29, wherein step b) comprisesthe synthesis of the at least two harmonized RNA species in one batch.33. The method according to claim 29, wherein step b) comprises an invitro transcription step.
 34. (canceled)
 35. The method according toclaim 29, wherein at least one RNA species comprises at least 500nucleotides.
 36. (canceled)
 37. The method according to claim 35,wherein at least one RNA species is a mRNA.
 38. The method according toclaim 37, wherein at least one RNA species comprises a 5′-cap structure.39-50. (canceled)
 51. The method according to claim 38, wherein at leastone RNA species comprises, in 5′ to 3′ direction, the followingelements: a) a 5′-cap structure b) optionally, a 5′-UTR element, c) atleast one coding region; d) a 3′-UTR element, e) optionally, a poly(A)sequence, preferably comprising 10 to 200, 10 to 100, 40 to 80 or 50 to70 adenine nucleotides, f) optionally, a poly(C) sequence, preferablycomprising 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosinenucleotides, g) optionally, a histone stem-loop, h) optionally, anadditional poly(A) sequence, or a fragment or variant of any one ofthese nucleic acid sequences. 52-54. (canceled)
 55. Compositioncomprising at least two RNA species, wherein the sequence of at leastone RNA species is adapted by altering the number of A and/or Unucleotides in the sequence of the at least one RNA species with respectto the number of A and/or U nucleotides in the original RNA sequence.56-58. (canceled)
 59. The method according to claim 38, wherein at leastone RNA species encodes Influenza virus hemagglutinin (HA) and/orInfluenza virus neuraminidase (NA), or a fragment or variant thereof.60-66. (canceled)
 67. Kit or kit of parts comprising the compositionaccording to claim
 55. 68. (canceled)
 69. Method of treating orpreventing a disorder, wherein the method comprises administering to asubject in need thereof a therapeutically effective amount of thecomposition according to claim
 55. 70. (canceled)
 71. The methodaccording to claim 38, wherein at least one RNA species is an mRNAcomprising a coding region, wherein the coding region has an increasedG/C content compared to the G/C content of an original coding sequence,wherein the encoded amino acid sequence is not modified compared to theamino acid sequence encoded by the corresponding original mRNA.
 72. Themethod according to claim 38, wherein the method is applied to at leastthree RNA species.
 73. The method according to claim 72, wherein the atleast three RNA species encode different influenza HA antigens.
 74. Themethod according to claim 29, further comprising: c) analysing the atleast two harmonized RNA species by chromatography.
 75. The methodaccording to claim 74, wherein the chromatography comprises HPLC. 76.The method according to claim 75, wherein the chromatography comprisesreversed phase HPLC.
 77. The method according to claim 76, wherein thereversed phase HPLC is with a column that comprises a porous material,selected from the group consisting of polystyrene, a non-alkylatedpolystyrene, an alkylated polystyrene, a polystyrenedivinylbenzene, anon-alkylated polystyrenedivinylbenzene, an alkylatedpolystyrenedivinylbenzene, a silica gel, a silica gel modified withnon-polar residues, a silica gel modified with alkyl containingresidues, selected from butyl-, octyl and/or octadecyl containingresidues, a silica gel modified with phenylic residues, and apolymethacrylate.